In vitro nematocidal activity of Punica granatum L. against gastrointestinal helminths in goats

Abstract

The objective of this study was to evaluate the in vitro ovicidal activity, phytochemistry, and toxicity of a saline extract obtained from peel of Punica granatum L fruits. The ovicidal activity was evaluated by the hatching inhibition of eggs recovered from fecal samples of naturally infected goats; the phytochemical analysis was carried out using the fruit peel; and the toxicity was tested on Artemia salina, using saline extract. The results showed that the ovicidal effect of the tested extract was 99% (25 mg mL⁻¹), 99% (12.5 mg mL⁻¹), 98% (6.25 mg mL⁻¹), and 95% (3.12 mg mL⁻¹), higher than that of the control drug, thiabendazole (83%). The phytochemical analysis showed presence of phenols, anthraquinones, and condensed and hydrolysable tannins in the fruit extract. The toxicity test of the extract of P. granatum showed an LC50 of 6.19 mg mL⁻¹, which indicates a safe use for a concentration of 3.12 mg mL⁻¹, since it was the tested concentration that was below the reliable LC50. The saline extract from peels of P. granatum has ovicidal activity, important secondary metabolites, and absence of toxicity at the lowest concentration tested. However, in vivo tests in experimental models are recommended before performing experiments in ruminants.

Introduction

The rearing of small ruminant animals is profitable and an important livestock agribusiness activity in Brazil (Davis et al. 2017), with an estimated herd of 9.5 million animals, which are responsible for the production of more than 14 thousand liters of milk per year; most of this herd is in the Northeast region of the country (IBGE, 2017). However, inadequate management practices and weather conditions of semiarid regions improve the development of endoparasites (Idris et al. 2019). This may result in low animal yields due to symptoms of infections that can lead to the death of the animals (Calvete et al. 2014; Idris et al. 2019).

Allopathic anthelmintics is the most parasitic disease control used (Maciel et al. 2014; Jiao et al. 2019). However, excessive use of these compounds contributed to the emergence of resistant strains of parasites (Fiel et al. 2017; Dey et al. 2020; Zajac and Garza 2020). This started a public health conflict due to residues in animal products and by-products consumed by humans. In addition, these contaminants are disposed in the environment together with excrements of treated animals, resulting in accumulation of these compounds in environmental waters and sediments (Cooke et al. 2017; Li et al. 2020; Wu et al. 2021).

Considering these issues, the use of phytotherapy may be viable for the control of gastrointestinal parasitosis (Davulure et al. 2020), since plants have unique proprieties and produce a wide variety of secondary metabolites (Idris et al. 2019). Moreover, they are available in nature and may have low toxicity (Lima et al. 2019), which can contribute to reduce chemical residues in the environment (Stucki et al. 2019).

Plants of the species Punica granatum L. (Punicaceae), known as pomegranate, stand out among these plants; it is native to Asia and widespread throughout the world (Oliveira et al. 2010; Di Stefano et al. 2019). This plant is used since 1550 BC to control parasitic diseases in ancient Egypt (Oliveira et al. 2010) and has been studied due to its many biological activities, such as anthelmintic, anti-inflammatory, antioxidant, immunomodulator, anticancer, and against skin and mucous infections, diseases related to the gastrointestinal tract, bacterial diarrhea, fungal infections, viruses, and respiratory diseases (Oliveira et al. 2010; Turrini et al. 2015; Varghese et al. 2017; Wong et al. 2021). According to Silva et al. (2018), protein fractions of the saline extract of Cassia fistula showed satisfactory anthelmintic effect (larvicide and ovicide). However, no studies about control of gastrointestinal parasites in small ruminants had been carried out using saline extract from peels of P. granatum fruits.

Thus, the objective of this study was to evaluate the in vitro ovicidal activity, phytochemistry, and toxicity of the saline extract from peels of P. granatum fruits for the development of a bioproduct that is effective against worms that affect small ruminants and that does not harm the environment.

Materials and methods

Description of the area and identification of plant species

The research was conducted in Mossoró, state of Rio Grande do Norte, Northeast region of Brazil (05°11′ 16.8″ S, 37° 20′ 38.4″ W, and altitude of 16 m), which is in the West Potiguar mesoregion and covers an area of 2100 km². The study area has a predominant semi-arid climate, with an average temperature of 27.4 °C and an irregular rainfall regime (Araújo et al. 2012).

The taxonomic identification of the species was based on the comparison between a specimen of the plant collected in Mossoró and a specimen form the collection of the Dárdano de Andrade-Lima Herbarium of the Center for Biological and Health Sciences of the Universidade Federal Rural do Semi-Árido (UFERSA). The identification of the exsiccate number 14,989 MOSS was obtained and registered at the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under the certificate AB92790.

Obtaining of the crude saline extract

The extract was obtained from peels of fruits of Punica granatum L. plants collected in a residential area, in Mossoró. The fruit peels were washed, dried at room temperature, and crushed. The obtained flour was subjected to 10% (w v⁻¹) extraction in a 0.15 M NaCl solution under stirring for 14 h at room temperature. The material was then filtered and subjected to centrifugation (4 °C; 8,000 rpm; 20 min) to obtain the supernatant corresponding to the crude saline extract (Nelson and Cox 2014). The 10% crude extract (25 mg mL⁻¹) was subjected to serial dilutions to obtain the concentrations of 12.5 mg mL⁻¹, 6.25 mg mL⁻¹, and 3.12 mg mL⁻¹.

Collection of fecal samples

The samples of feces were collected directly from the rectal ampoule of goats. The egg count (eggs per gram of feces—EPG) was carried out using 4 g of feces, according to the technique used by Gordon and Whitlock (1939) and Chagas et al. (2011), in quintuplicate. The five samples were collected from animals infected, with an average fecal pool of 2000 eggs per gram of feces in the EPG count. The samples were collected from animals that presented the end of the residual period of 90 days with no treatment with synthetic chemical (Niciura et al. 2009). The goats were randomly selected, regardless of race or sex.

Five stool cultures were carried out referring to the sample pool of each collection, according to the cultivation technique described by Roberts and O’Sullivan (1950) and Chagas et al. (2011). Where approximately 20–30 g of feces were cultivated in a wide-mouth flask, with humidification performed daily by spraying and aeration provided by semi-closing the flask with a Petri dish and the aid of a paper shield at the edge. The culture was left to rest for seven consecutive days. To recover the larvae (L3), the flask was uncapped and water was added to the culture up to the edge of the flask, placing a Petri dish in the mouth of the flask and abruptly pouring the set. After 6 h, the larvae were collected, with the aid of a Pasteur pipette, from the liquid that drained from the flask and kept in refrigeration. The identification took place by verifying the morphological characteristics of the larvae using a specific key (Ueno and Gonçalves 1998, Van Wry et al. 2004), where the first 100 larvae were identified for each stool culture. With identification of the genera Haemonchus spp., Oesophagostomum spp., Strongyloides spp., and Trichostrongylus spp.

Egg hatch test (EHT)

The recovery of nematode eggs was carried out five times, after each fecal sampling, according to the methodology described by Hubert and Kerboeuf (1992). Five egg hatch test (EHT) were carried out according to the methodology described by Coles et al. (2006). On average, 100 eggs per wells (calculated within 100 µL) was added to 24 wells plates, using five wells per treatment. For negative controls 100 µL of recovered eggs and 400 µL of 0.15 M NaCl were added; for positive controls with 100 µL of the recovered fluid containing eggs and 400 µL of thiabendazole (32 µg mL⁻¹) were added. In the experimental group, 100 µL of recovered eggs and 400 µL of saline extract (25 mg mL⁻¹), 100 µL of recovered eggs and 400 µL of saline extract (12.5 mg mL⁻¹), 100 µL of recovered eggs and 400 µL of saline extract (6.25 mg mL⁻¹), and 100 µL of recovered eggs and 400 µL of saline extract (3.12 mg mL⁻¹). Each tested concentration and positive and negative controls were performed in quintuplicate. The plates were incubated in biochemical oxygen demand (BOD) for 48 h at temperature of 27 °C and controlled humidity, with subsequent addition of Lugol's solution and counting of eggs and larvae in each wells, using an inverted microscope.

Phytochemical analysis

Peels of P. granatum fruits were dried at room temperature and crushed; the obtained flour was subjected to analysis of secondary chemical metabolites, based on the methodology used by Matos (2008). Secondary metabolite classes were identified by observing the combination and colorimetric and fluorescence reactions. Reactions were carried out to identify alkaloids (Dragendorff, Mayer and Bertand test), anthraquinones (Borträger test), coumarins (fluorescence test), phenols (ferric chloride reaction), flavonoids (cyanidin or Shinoda reaction), steroidal nuclei (Liebermann-Burchard test), triterpene nuclei (Salkowski test), saponins (foam test—vigorous form), condensed and hydrolysable tannins (Stiasny test), and free tannins (gelatin).

In vitro acute toxicity analysis

The evaluation followed the protocol described by Rodriguez et al. (2009); the hatching of A. salina nauplii (obtained commercially) eggs was carried out in 1 L of distilled water containing 18 g of NaCl and 5 g of NaHCO₃, under constant lighting and aeration, with incubation for 48 h. Ten nauplii were transferred to wells in 24 wells plates. Five wells containing 100 µL of A. salina culture solution and 400 µL of saline extract (25 mg mL⁻¹), five wells containing 100 µL of A. salina culture solution and 400 µL of saline extract (12.5 mg mL⁻¹), five wells containing 100 µL of A. salina culture solution and 400 µL saline extract (6.25 mg mL⁻¹), and five wells containing 100 µL of A. salina culture solution and 400 µL saline extract (3.12 mg mL⁻¹) in each wells. This test was performed in quintuplicate.

Data analysis

The data of the eggs and larvae counts in the EHT and of the toxicity test were tabulated in a spreadsheet and subjected to data normality test, and the results of the 50% lethal concentration (LC50) test in the toxicological assay with A. salina were calculated by non-linear regression, using the GraphPad Prism 6.0 program. The eggs hatching inhibition percentage of each experimental group was obtained by the equation:

$$\text{Hatching}\text{inhibition}\text{percentage}=\text{number}\text{of}\text{eggs}/\left(\text{number}\text{of}\text{eggs}+\text{number}\text{of}\text{first}\text{stage}\text{larvae}\right)\times 100$$

The results of the tested concentrations were expressed as eggs hatching inhibition percentage and the differences between these percentages were determined by the Tukey's test at p < 0.05.

Results

The saline extract showed greater eggs hatching inhibition percentage when using the concentrations of 25 mg mL⁻¹ (99%) and 12.5 mg mL⁻¹ (99%). The concentration of 6.25 mg mL⁻¹ showed a 98% inhibition, and the lowest concentration (3.12 mg mL⁻¹) a 95% inhibition. The positive control showed an inhibition of 85% and the negative control showed an inhibition of 17%, with no significant effect on the eggs hatching. However, the eggs hatching of 83% in the negative control shows the viability and that the incubation time and conditions were satisfactory for the eggs hatching.

The phytochemical test using peels of P. granatum fruits showed the presence of phenols, anthraquinones, condensed tannins, and hydrolysable tannins. The acute toxicity test showed the LC50 at the concentration of 2.477% (6.19 mg mL⁻¹). Therefore, the concentrations of 25 mg mL⁻¹, 12.5 mg mL⁻¹, and 6.26 mg mL⁻¹ are toxic to A. salina, and the concentration of 3.12 mg mL⁻¹ showed an eggs hatching inhibition percentage of 95% with no acute toxicity for the tested organisms.

Discussion

All tested concentrations of the saline extract showed ovicidal efficacy against gastrointestinal parasites in goats, with no statistically significant difference from the positive control. The ovicidal activity of the positive control (83%) denotes that the eggs tested were from parasites with anthelmintic resistance. According to Coles et al. (2006) eggs counts below 90% may be caused by presence of parasitic resistance, denoting the importance of the activity of the tested plant drug.

The antiparasitic efficacy of the extract may be related to the secondary metabolites found, mainly phenols, which are a class of phenolic compounds (Delgado et al. 2019) characterized by an antioxidant action capable of eliminate free radicals (Pilerood and Prakash 2014) and increase the animal immunity (Sprenger et al. 2015). Phenols can affect the decoupling of the oxidative phosphorylation responsible for ATP production, interfering with the energy production, which is necessary for development and survival of the parasite (Salhan et al. 2011).

Tannins are other metabolites found in the extract, which are generally classified into condensed tannins (polyphenolic compounds based on flavonoids) and hydrolysable tannins (compounds that undergo hydrolytic cleavage) (Laurichesse and Avérous 2014). The ovicidal mechanism of action may be related to their synergistic interaction with enzymes, such as lipases, proteases, and aminopeptidases. Inhibition of enzymes responsible for the hatching can interfere with the eggshell disintegration process, inhibiting the eggs hatching (Molan and Faraj 2010). Tannins can interact with other metabolites, increasing cell permeability, which favors their interaction with free proteins or cuticle glycoproteins of parasites, hindering nutrient absorption, mobility, reproduction and, consequently, causing their death (D'addabbo et al. 2011; Cala et al. 2012; Botura et al. 2013).

Studies have shown that methanol extract from peels of P.granatum fruits reduce the larvae motility (Jabeen et al. 2015) and eggs hatching rate of parasites of the species Haemonchus contortus (Ahmed et al. 2020). These results were attributed to the action of the extract secondary metabolites found in the phytochemical analysis, mainly tannins. Anthraquinones, which were also found in those studies, are polyphenolic compounds present in several plants, which present strong color (Dufossé, 2014). Natural anthraquinones have high bioactivity, such as antioxidant, diuretic, purgative, anti-inflammatory, laxative, vasorelaxant, antimicrobial, and immunosuppressive (Duval et al. 2016). Their action may be related to their capacity to inhibit cell development by different mechanisms, such as apoptosis induction, intercalation and binding with DNA, and inhibition of the enzyme topoisomerase (Balachandran et al. 2016; Pérez-Pertejo et al. 2019). Anthraquinones also present antibacterial activity, predominantly by inhibiting or interfering with redox processes inside the cell, which indicates that this compound can be diffused through the membrane of bacteria (Chan et al. 2011). This effect is not ruled out for parasites.

Regarding the toxicity, the concentrations of 3.12 mg mL⁻¹ presented 95% inhibition and was not considered toxic. Therefore, researches using toxicity bioindicators for acute and chronic in vitro tests are important and necessary to investigate toxicity of natural compounds, mainly before in vivo tests (Cansian et al. 2017).

A research using ethanolic, hexanic, chloroformic extracts, and ethyl acetate from seeds of Artocarpus heterophyllus subjected to acute toxicity tests with A. salina showed that samples of the hexanic extract proved to be toxic to these organisms, with an LC50 of 50.14 µg mL⁻¹ (Burci et al. 2019). Costa et al. (2015) conducted toxicity tests using aqueous extract from leaves of Phthirusa pyrifolia, which is rich in phenolic compounds and tannins, for different organisms, including A. salina, and obtained an LC50 of 17.19 mg mL⁻¹. Tanamatayarat (2016) evaluated the toxicity of Protium serratum against nauplii of A. salina and found that the essential oil (ethanolic extract) from its fruits (rich in tannins) has high toxicity, with an LC50 of 3.57 µg mL⁻¹. In addition, AL-Saeedi et al. (2017) evaluated the cytotoxic activity of Ziziphus jujube, which is rich in secondary metabolites, including tannins, against A. salina and found that the crude leaf extract (using butanol as solvent) showed an LC50 of 8.685 µg mL⁻¹ and the crude fruit extract (using ethyl acetate as solvent) showed an LC50 of 11.617 µg mL⁻¹.

The extract of fruit peels of P. granatum proved to have in vitro biological activity. The presence of metabolites presenting mechanisms of action, as described above, were identified by the phytochemical test. The toxicity test showed that low concentrations of the extract are not toxic to A. salina. Therefore, this study showed that P. granatum plants can be a viable alternative for further in vitro and in vivo studies using experimental models and, later, using ruminant animals. Moreover, this plant species can be used at the concentrations that presented no in vitro toxicity to bioindicator organisms. This would contribute to environmental conservation, since it is expected that, after further tests, it can be used to reduce environmental contamination by toxic residues present in excrement of treated animals and decrease the use of chemical anthelmintics and parasitic resistance.

Authors' contributions

RCSF and ACDSB conceived and designed the research. RCSF, TNB, CKOR, and JILB conducted the experiments. MDCS and KMPS contributed with new reagents and analytical tools. HPM and JCSP analyzed the data. RCSF and ACDSB wrote the manuscript. All authors read and approved the manuscript.

Funding

The authors did not receive support from any organization for the submitted work.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

The study was carried out according to ethical and legal precepts, approved by the Animal Ethics Committee of the “UNIVERSIDADE FEDERAL RURAL DO SEMI-ÁRIDO” (CEUA-UFERSA) (23091.009318/2016-40).