1. Introduction
In most developing countries, livestock production is an essential source of income and contributes to food security. Indeed, Livestock farming is an important source of income for those involved in the livestock sector, and also contributes to the fight against malnutrition by providing protein and micronutrients to the population.
However, gastrointestinal strongylosis in ruminants is one of the main diseases that cause substantial economic losses to livestock keepers [1] . Particularly in small ruminants, infestations due to gastrointestinal nematodes constitute a fundamental constraint in breeding [2] [3] because they cause growth retardation and weight loss in these animals [4] [5] . Thus, the parasitic fauna of the digestive tract of small ruminants is dominated by strongyles, particularly Haemonchus contortus (H. contortus), by its frequency and intensity [6] [7] [8] [9] . Control of these parasites has long relied on the use of synthetic anthelmintics for their efficacy.
In many developing countries, small-scale livestock keepers are poor, so they have difficulty accessing veterinary products due to their high cost [10] [11] . Synthetic anthelmintics administered to animals are eliminated directly into nature in the form of active metabolites, via urine and faeces, constituting a source of environmental contamination that is potentially toxic for biodiversity. Globally, the use of reference anthelmintics is at the root of the development of chemoresistance [12] . To counteract the inaccessibility and chemoresistance associated with reference anthelmintics, emphasis should be placed on the use of plant resources that are available and known to farmers to solve the problems posed by these diseases. Thus, medicinal plants could provide a therapeutic response adapted to populations’ financial means and socio-cultural environment.
Medicinal plants are a group of great socio-economic importance because they contain active components for treating various diseases [13] . This is why the middle and poor strata of Africa and the world often resort to certain plants with therapeutic virtues to treat or alleviate certain diseases [14] [15] [16] .
In Burkina Faso, some researchers have already shown interest in plants with anthelmintic properties for veterinary use that could be an alternative to synthetic anthelmintics. These include: Balanites aegyptiaca [17] , Saba senegalensis [18] , Cassia sieberiana, Guiera senegalensis and Excoecaria grahamii [19] , Securidaca longepedunculata [20] . For more efficiency in improving the health of small ruminants, it would be wise to conduct studies on forage plants for their nutritional values and also for their richness in specific secondary metabolites such as tannins, flavonoids, saponins and alkaloids, whose anthelmintic properties and effects on zootechnical performance are known [21] [22] [23] . Burkinabe traditional medicine has several medicinal plants used by the population to treat gastrointestinal parasitosis. These include Gomphrena serrata L. (Amaranthaceae) [24] . Research on forage plants with anthelmintic properties will have the advantage of revealing the levels of primary and secondary metabolites with anthelmintic action contained in these plants.
For our part, the study of Gomphrena serrata (G. serrata) deserves to be conducted in this sense because this plant has the advantage of being a forage plant and containing bioactive substances such as flavonoids, saponins, tannins, alkaloids [25] . This study aims to evaluate, in vitro, the anthelmintic effect of three extracts of G. serrata on the inhibition of egg hatching and the inhibition of the motility of adult worms of Haemonchus contortus.
2. Materials and Methods
2.1. Animal Material
The biological material consists of animal material consisting of adult worms of Haemonchus contortus and.
2.2. Plant Material
Plant material consists of the G. serrata plant. The plants were collected in July 2022, near the city of Ouagadougou, in the morning between 6 am and 8 am and identified by the taxonomists of the Herbarium of the UFR/SVT of the Joseph KI-ZERBO University. G. serrata L. was already identified in 2017 under the numbers: N˚ ID: 17012; N˚ Echantillon: 6848 du 08/07/2017, of which a sample of the species was kept in the Herbarium of the Plant Biology and Ecology Laboratory of the Department of Plant Biology and Physiology (LaBEV). The collected plants were washed with tap water and dried in the shade at room temperature in the laboratory between 25 and 28°C. Once dried, the plants were ground to powder in an electric grinder. The powder thus obtained was used to prepare three extracts: an extract by aqueous maceration, an extract by aqueous decoction, and an extract by hydroethanolic maceration. These extracts were freeze-dried before being used for the various tests.
2.3. Preparation of Extracts
The method used is the solid-liquid extraction method. Plant powder, representing the solute, is the solid phase; distilled water (or distilled water and ethanol), representing the solvent, is the liquid phase.
2.3.1. Aqueous Decoction
A 100 g test portion of the G. serrata plant powder was placed in a 2000 mL Erlenmeyer flask. A volume of 1000 mL of distilled water was added to the test portion due to the mucilaginous characteristic of the plant powder. After homogenisation with a glass rod, the mixture of plant material and extraction solvent was boiled on a hot plate for 1 h. After cooling, the extract was filtered through a fine mesh nylon cloth. The filtrate obtained was centrifuged at 2000 rpm for 10 min, and the supernatant was collected and frozen at −24°C for freeze-drying. The resulting lyophilisate was weighed to determine the extraction yield, represented by the mass of extracted material in 100 g of powder, and then stored in a refrigerator at 4°C.
2.3.2. Aqueous Maceration
A 100 g test portion of the G. serrata plant powder was placed in a 2000 mL Erlenmeyer flask. 500 mL of distilled water was added to the test portion. After homogenisation with a glass rod, the mixture of plant material and solvent extractor was kept at room temperature in the laboratory (30 °C) for 24 h. The extract was stirred from time to time with a glass rod. After a 24 h stay, the extract was well homogenised, filtered through a fine mesh nylon cloth, and pressed. The filtrate obtained was then centrifuged at 2000 rpm for 10 min. The resulting supernatant was collected, frozen at −24°C, and then lyophilised. The resulting lyophilisate was weighed to determine the extraction yield, represented by the mass of extracted material in 100 g of powder, and then stored in a refrigerator at 4°C.
2.3.3. Hydroethanolic Maceration
A 100 g test portion of the G. serrata plant powder was placed in a 2000 mL glass jar with a screw lid. To the test portion, 800 mL of a mixture of ethanol and distilled water in the ratio of 70:30; (v/v) was added. The plant material and extractor solvent mixture was homogenised using a glass rod and then kept at the laboratory’s ambient temperature (30°C) for 48 h. The extract was then filtered through Wattman No. 5 paper. The residual pomace was depleted with portions of the extracting solvent until a faded filtrate was obtained. The hydroalcoholic extract of the sample was then concentrated to ¾ under reduced pressure in the rotavapor. The resulting concentrated extract was frozen at −24°C and then lyophilised. The resulting lyophilisate was weighed to determine the extraction yield, represented by the mass of extracted material in 100 g of powder, and then stored in a refrigerator at 4°C.
2.4. Phytochemical Screening
The phytochemical screening aimed to determine the groups of bioactive chemicals in the aqueous and hydroalcoholic extracts of G. serrata. The characterisation tests of the different groups of substances were carried out in a liquid medium with chemical reagents specific to each group of bioactive substances. The method used was the one described by Ciulei, adapted by the chemistry laboratory of the Research Institute for Health Sciences (IRSS) [26] . The principle of this method is based on the ability of the functional chemical groups of bioactive substances to react with specific chemical reagents to give their characteristic colored products. The FeCl3 test has identified tannins (polyphenols); flavonoids by the Shibata reaction or cyanidin test; saponins by the foam test; steroids and triterpenes by the Liebermann-Burchard test; alkaloids by the Mayer and Dragendorff tests; Anthracenosides by Bornträger’s test; Coumarins and derivatives by Feigl’s test; Cardenolides by Kéddé’s reagent; Reducing compounds by Fehling’s test; Anthocyanosides by Bate Smith’s reagent.
2.5. Acute Toxicity Assay
The toxicity assay was carried out following the Organization for Economic Co-operation and Development (OECD) test guideline 423 concerning acute oral toxicity [27] . Before administration of the G. serrata extracts, the female rats were divided into batches of 3 per cage and fasted for 4 h without food but with free access to water. For each extract, the test was conducted in two stages. In the first phase, 3 rats each received a single oral dose of 2000 mg/kg b.w of each extract. After administration of the extracts, the animals were observed every 30 min for 2 h, after which water and food were re-introduced. The rats were further observed once a day for up to 14 days following treatment for probable signs of toxicity, such as behavioral or physiological changes and death and the latency of death [28] [29] . All the animals were weighed before the start of the test and then weighed at D1, D2, D3, D7 and D14 during the test period. After 14 days, the animals were anaesthetised with ketamine (150 mg/kg) and sacrificed. Vital organs such as the heart, lungs, kidneys, spleen, and liver were removed, dissected, and weighed. The relative weight of each organ was calculated.
2.6. Anthelmintic Assays
2.6.1. Collection of H. contortus Eggs
Adult female worms are collected, placed in PBS, and then slightly diluted in a mortar with a porcelain pestle to release the eggs. The resulting solution is filtered through mesh screens of different sizes (1 mm and 100 μm). Another 38 μm mesh screen is then used to retain the released eggs. The released eggs are recovered by rinsing the sieve with distilled water. The resulting egg suspension was adjusted to approximately 1000 eggs per mL.
2.6.2. Eggs Hatching Essay
100 uL of the egg suspension adjusted to 1000 eggs/mL was distributed to the wells of a 24-well plate. Each well added a volume of 1900 μL of extract of increasing concentration to 0.125; 0.25; 0.5; 0.625; 1; 2; 3 mg/mL. Albendazole was used as a positive control at the concentrations 0.0019; 0.00780; 0.03125; 0.06250; 0.125; 0.25 and 0.5 mg/mL. Distilled water was used as a negative control. The plate was resealed and incubated at 25°C for 48 hours. After incubation, a drop of formalin was added to each well to stop the evolution of the eggs. The contents of each well were then placed on a microscope slide, and the number of developed larvae was assessed under a light microscope (Gx40). The test was repeated three times at different concentrations for each of the extracts and controls. The inhibitory activity of the plant extracts was determined by calculating the egg hatch inhibition rate (EHI) using the formula described [30] :
2.6.3. Collection of Adult Worms
After slaughtering sheep or goats naturally infested with H. contortus, the abomasums were collected and packed in a cooler. In the parasitology laboratory, each abomasum was incised longitudinally in order to release the reddish coloured adult worms due to their hematophagous feeding mode. The worms were then carefully collected and placed in a petri dish containing a physiological Phosphate Buffer Saline (PBS, pH: 7.02) at 37°C.
2.6.4. Adult Worms Mortality Assay
Test extract solutions are prepared with PBS in six concentrations 0.75; 1; 1.25; 1.5; 2; 3; 4 mg/mL to perform the test [31] . Collected worms with good motility were placed in Petri dishes at a rate of three worms per dish, in a total volume of 8mL of each test extract prepared with the PBS solution and placed in an incubator at 35°C. A negative control (PBS buffer) and a positive control levamisole 0.0625; 0.125; 0.25; 0.5; 1; 2 and 2.5 mg/mL, prepared in PBS are used. The test is repeated three times for each concentration and the controls. The inhibition of adult worm motility in the treatments performed is used as the criterion for anthelmintic activity. After the worms were exposed to the extracts, motility was observed under a magnifying glass every 6, 20, and 24 hour and the number of dead worms was assessed at each observation. Any worm showing continuous total immobility for 10 seconds is suspected to be dead. It is then carefully removed from the well and placed in 10 ml of PBS solution for possible revitalisation. If the worm regains vitality within 30 minutes of removal from the test well, it is considered alive and returned to its well, and observations continue. If not, the worm is declared dead. The mortality rate per concentration and type of extract is calculated using the formula:
2.7. Statistical Analysis
Excel spreadsheet software obtained means, standard deviations, and illustrative graphs. Comparisons of treatments and analysis of the significance of a dose effect and a solvent effect are performed using GraphPad prism. The effects of the different doses on the motility of adult worms were compared using Student’s t-test.
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Table 1. The yield of G. serrata extracts.
GS_AD = G. serrata Aqueous Decoction; GS_AM = G. serrata Aqueous macerate; GS_HEM = G. serrata Hydroethanolic macerate
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Table 2. Phytochemical groups found in G. serrata extracts.
3. Results
3.1. Extraction Yields
The extraction yields varied according to the solvents and the extraction methods (Table 1).
3.2. Phytochemical Screening
Phytochemical analysis (colorimetric reactions) revealed the presence of tannins, saponoside, reducing compounds, coumarins and derivatives, anthocyanosides, steroids, triterpenes, and flavonoids (Table 2).
3.3. Acute Toxicity Assay
In acute oral toxicity assay, after 14 days of observation post-treatment, no behavioral changes such as motor activity, breathlessness, alertness, restlessness, diarrhea, tremor, convulsion and coma were observed at the administered doses of AD, AM and HEM of G. Serrata. Also, the rats were physically active, and no death was observed in different experimental animals groups in a single dose of 2000 mg/kg/b.w. of extracts.
However, the average body weights of all control animals and those treated with each of the three extracts showed a relative increase over the 14 days (Figure 1). There was a slight upward trend in the body weights of homologous
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Figure 1. Evolution of the weight of the female rats in the control group in comparison with those in the experimental groups.
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Table 3. Changes in the relative weights of female rat organs in the toxicity test.
groups of mice treated under the same experimental conditions. These changes in body weight were not statistically significant.
Macroscopic examination of fresh vital organs such as the heart, lungs, liver, kidneys and spleen of control animals and those treated with hydroethanolic macerate, aqueous decoctate and aqueous macerate shows that there was no change in the colour or appearance of these various organs. The relative organ weights of control rats and those treated with 2000 mg/kg bw of the three extracts are given in Table 3.
With n = 6; analysis was made with Two-way ANOVA followed by “Dunnett” multiple comparisons test. No statistically significant difference between the control group and the different extracts (p > 0.05).
3.4. Eggs Hatching Inhibition
The egg hatching inhibition in vitro of G. serrata extracts were compared to the albendazole used as a positive control (Figure 2).
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Figure 2. Effects of the different concentrations of the three extracts of G. serrata on H. contortus eggs hatching.
n = 6 ** p < 0.01, *** p < 0.001 GS_AD vs. GS_HEM, ### p < 0.001 GS_HEM vs. GS_AM, λ p < 0.1, λλλ p < 0.001 GS_AD vs. GS_AM is considered significant within each row, compare columns. Analysis was made with Two-way ANOVA followed by Tukey’s multiple comparisons test. p < 0.05 significant between extracts. No significant difference between the different extracts (p > 0.05).
The 50% inhibitory concentrations (IC50) were: GS_AD = 0.5454; GS_HEM = 0.4572; GS_AM = 0.5760 and Abz = 0.03437.
After 48 hours of contact between the eggs of H. contortus and the different concentrations of the three extracts of G. serrata, the hatching inhibition rate was 100% for the three extracts from 3 mg/mL.
3.5. Adults Worms Mortality
The motility of adult H. contortus worms was inhibited in vitro by G. serrata extracts compared to the positive control, levamisole (Figure 3). The HEM has proven to be more effective than the AD, which is, in turn, more effective than the AM.
n = 6 ** p < 0.01, *** p < 0.001 GS_HEM vs. GS_AM, ## p < 0.01 GS_AD vs. GS_AM is considered significant compared each row, to columns. Analysis was made with Two-way ANOVA followed by Tukey’s multiple comparisons test. p < 0.05 significant between extracts. There is no significant difference between the different extracts (p > 0.05). After 24 hours of contact between the adult worms, the mortality rate reaches 100% for the three 4 mg/mL extracts.
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Figure 3. Effects of the different concentrations of the three extracts of Gomphrena serrata on the adult worms of H. contortus.
The 50% lethal concentrations (LC50) were: GS_AD = 1.418; GS_HEM = 1.329; GS_AM = 1.652 and Lev = 0.2753.
4. Discussion
In the phytochemical study, the highest yields were observed with the aqueous decoction at 21.03%, followed by the aqueous macerate at 17.42%. The lowest extraction yield was with hydroalcoholic macerate: 13.95%. The extraction yield, therefore, depends on the nature and physicochemical characteristics of the solvents used, and in particular their polarity, and also on the solubility of the substances contained in the plant material. The higher yield of decoction may be due to the action of heat, a physical catalyst that dilates cell membranes, promoting the escape of hydrophilic compounds and also certain emulsified hydrophobic compounds.
Phytochemical screening reveals the presence of tannins, saponins, reducing compounds, coumarins and derivatives, steroids and triterpenes, and flavonoids at the level of the three extracts. However, anthocyanosides are only found in the aqueous decoction and maceration. The absence of anthocyanosides in the hydroalcoholic macerated could be explained by a lack of solubility and affinity with the majority extracting solvents, ethanol. Indeed, the absence of anthocyanins in the ethanolic extract of the leaves of Myrtus communis and Rhamnus alaternus [32] . Cardenolides and alkaloids are absent in all three extracts. Identifying tannins, saponins, steroids and triterpenes as flavonoids in G. serrata agrees with the work of Prasanna et al. [33] . He also highlighted the alkaloids. Our extracts have the advantage of containing coumarins which other publications on G. serrata have not highlighted. The plant development stage could explain phytochemical composition differences at harvest and climatic conditions, edaphic conditions, and genetic factors [34] [35] .
The acute toxicity study tests the harmful effects of an agent on the organism after a single or short-term exposure [36] [37] . Assessment of acute systemic toxicity in rats given a lethal dose of 2000 mg per kg body weight of each extract resulted in no deaths or signs of toxicity after 14 days of follow-up. According to the acute toxicity class method of OECD test guideline 423 and the Global Harmonized System, AD, AM, and HEM extracts of G. Serrata can be classified to the 5th toxicity class [27] [38] . Therefore, the LD50 value was estimated at 5000 mg/kg body weight, and the extracts have a relatively low acute oral toxicity.
Regarding the in vitro inhibition of eggs hatching, our results show an inhibition rate of 100% from 3 mg/mL for the three types of extracts. The mortality rate of adult worms is 100% for three extracts: AD, AM and HEM, at a concentration of 4 mg/mL in 24 hours. This indicates that the three extracts effectively inhibit the motility of adult worms. However, our extracts are less effective in inhibiting adult worms’ motility than levamisole, which gives a mortality rate of 100% in 24 hours with a 2.5 mg/mL concentration. Our three extracts are less effective in the inhibition of egg hatching than albendazole, which gives a rate of 100% in 48 hours with a concentration of 0.5 mg/mL. HEM is more efficient on eggs and Haemonchus contortus adult worms compared to others with IC50 of 0.457 mg/mL and LC50 of 1.329 mg/mL, respectively. Our results are comparable to those obtained by Houngnimassoun et al. [23] , which is 100% from 64 mg/mL of Ficus exasperata, and those obtained by Belemlilga et al., who showed that the extract of Saba senegalensis gave a 97% hatch inhibition rate with 15 mg/mL [31] . G. serrata, through its three extracts, proved effective against the two parasitic stages considered in this study. However, the HEM extract was more effective than the AD and AM extracts. This suggests that the mode of extraction by HEM of G. serrata should be favored for effective anthelmintic action because of its ability to effectively extract polar and apolar compounds. The anthelmintic activity of G serrata would be due to secondary metabolites such as flavonoids, tannins, and saponins [39] [40] [41] . Indeed, tannins interfere with oxidative phosphorylation by blocking ATP synthesis in H. contortus [42] . Condensed tannins are the main compounds responsible for the anthelmintic effect [21] [41] . Inhibitions of worm motility and egg hatch are due to condensed tannins [43] , which would diffuse to the membrane surface of eggs and larvae to bind to free membrane proteins, thus inhibiting egg hatch [44] [45] . Also, the anthelmintic effect of saponins would be explained by the destabilization of the membranes, hence an increase in the permeability of the cells, which will cause the turgidity of the cells and their bursting [39] . Today’s challenge in the fight against helmintiasis is to use natural anthelmintics, to reduce the selection pressure of helminths by favouring the use of forage plants rich in flavonoids, tanninsand saponins, and by eliminating helminth-infected animals. The effectiveness of the effect observed in the anthelmintic effect would be due to a synergistic action of secondary metabolites such as flavonoids, tannins, and saponins. The in vivo anthelmintic properties of Gomphrena serrata need to be studied in order to confirm the efficacy of plant extracts on the different stages of Haemochus contortus development.
5. Conclusion
Our study first confirmed the richness of Gomphrena serrata in metabolites such as tannins, saponins, reducing compounds, coumarins and coumarin derivatives, anthocyanins, steroids, triterpenes, and flavonoids. Then, it showed that G. serrata inhibits the hatching of eggs and the motility of adult worms of Haemochus contortus. The HEM of Gomphrena serrata showed more efficacy than the AD and the AM, respectively, on the inhibition of egg hatching and the inhibition of the motility of adult Haemochus contortus worms. The traditional use of the leaves of Gomphrena serrata as an anthelmintic then seems to be justified. However, it would be necessary to carry out in vivo parasitological studies to consider the extracts’ metabolism in the animal’s digestive tract. The results of our study reveal the scientific basis of Gomphrena serrata anthelmintic activity, and will also enable us to enhance the value of this neglected plant.
Acknowledgements
The authors would like to thank the Laboratory for Research and Development of Phytomedicines and Medicines (LR-D/PM) of the Institut de Recherche en Sciences de la Santé (IRSS/CNRST), Ouagadougou, Burkina Faso.