Introduction
Small ruminant husbandry plays a vital role in the socio-economic aspect of developing nations, serving as an important source of sustenance and revenue. Furthermore, it offers promising opportunities for small and marginalised farmers while also meeting the escalating worldwide demand for meat (Moyo et al. Reference Moyo, Masika and Muchenje2013; Sujon et al. Reference Sujon, Mostofa, Jahan, Das and Rob2008). Unfortunately, gastrointestinal nematodosis (GIN) is a major constraint in the health and production potential of farmed animals. Nematodes of genera Haemonchus, Trichostrongylus, Ostertagia, Oesophagostomum, Bunostomum, Cooperia, Nematodirus, and Strongyloides are responsible for the clinical disease in small ruminants (Asmare et al. Reference Asmare, Sheferaw, Aragaw, Abera, Sibhat, Haile, Kiara, Szonyi, Skjerve and Wieland2016; Tayo et al. Reference Tayo, Poné, Komtangi, Yondo, Ngangout and Mbida2014). Amongst all, Haemonchus is highly pathogenic and responsible for causing anaemia, diarrhoea, weight loss, and mortality in heavy infection (Mortensen et al. Reference Mortensen, Williamson, Terrill, Kircher, Larsen and Kaplan2003). For controlling these nematodes, different classes of chemical compounds (anthelmintics) viz., benzimidazoles, imidazothiazoles, and macrocyclic lactones are used extensively in farmed animals (Kaplan Reference Kaplan2013; Kaplan and Vidyashankar Reference Kaplan and Vidyashankar2012). Extensive use of anthelmintics leads to development of anthelmintic-resistant worms, presence of drug residues in meat and milk, drug toxicity, etc. (Shalaby Reference Shalaby2013). The other integrative approaches involve use of ethno-veterinary medicines, selective breeding, grazing management, etc. (Mphahlele et al. Reference Mphahlele, Tsotetsi-Khambule, Moerane, Komape and Thekisoe2021; Wardynski Reference Wardynski2019).
Ethno-veterinary treatment like use of plants of medicinal value has been approached all over the world to combat anthelmintic resistance. Many reviews are available on the experimental studies carried out to evaluate the possible anthelmintic potential of several indigenous plants against GINs viz., Musa paradisiaca and Trianthema portulacastrum (Pakistan; Hussain et al. Reference Hussain, Khan, Iqbal, Sajid and Khan2011); Agave sisalana, Caesalpinia pyramidalis, Annona muricata (Brazil, Botura et al. 2013; Borges-dos-Santos et al. Reference Borges-dos-Santos, Lopez, Santos, Zacharias, David, David and Lima2012; Ferreira et al. Reference Ferreira, Castro, Chagas, França and Beleboni2013); Moringa oleifera (Cameroon, Tayo et al. Reference Tayo, Poné, Komtangi, Yondo, Ngangout and Mbida2014; Philippines, Cabardo and Portugaliza Reference Cabardo and Portugaliza2017); Entada leptostachya and Prosopis juliflora (Kenya, Mutembei et al. Reference Mutembei, Kareru, Njonge, Peter, Kutima, Karanja and Kimani2015); Carica pubescens (Indonesia, Baihaqi et al. Reference Baihaqi, Widiyono and Nurcahyo2020), etc. The herbal anthelmintics are organic, non-toxic, biodegradable, and do not leave any residue in animal products. Besides, due to the presence of various phytoconstituents with diverse mechanisms of action, the likelihood of developing resistance is minimal (Chagas Reference Chagas2015). Hence, plant species having anthelmintic efficacy were enclosed in the pharmacopoeias (Pathak and Chhabra Reference Pathak and Chhabra2014).
In India, infection by GINs in general and H. contortus in particular is being managed by extensive usage of anthelmintics, as there is no suitable vaccine available (Roeber et al. Reference Roeber, Jex and Gasser2013). Further, resistance in GINs against benzimidazole, levamisole, and ivermectin has been documented (Rathod et al. Reference Rathod, Kasaralikar, Ravindra, Sunilchandra, Halmandge and Patil2019; Singh et al. Reference Singh, Srivastava, Yadav, Singh and Singh2010; Vohra et al. Reference Vohra, Singh and Kumar2019; Yadav and Garg Reference Yadav and Garg2004). Researchers are also trying to identify potential plant-based dewormers for effective management of GINs (Singh et al. Reference Singh, Swarnkar, Khan and Bhagwan2008, Reference Singh, Swarnkar, Khan, Bhagwan and Dubey2011; Swarnkar et al. Reference Swarnkar, Singh, Khan and Bhagwan2009). There is literature on efficacy of the use of plant extracts like Embelia ribes (Swarnkar et al. Reference Swarnkar, Singh, Khan and Bhagwan2009), Azadirachta indica (Singh et al. Reference Singh, Swarnkar, Khan and Bhagwan2008), Andrographis paniculata (Singh et al. Reference Singh, Swarnkar, Khan, Bhagwan and Dubey2011), Butea monosperma (Singh et al. Reference Singh, Singh, Verma, Singh and Anand2015), Annona squamosa, and Nicotiana tabacum extracts (Dixit et al. Reference Dixit, Das, Dixit and Sharma2019) against GINs in various geographic regions of the Indian subcontinent. Based on a literature search for the anthelmintic property-bearing plants which are abundantly and easily found in India, Moringa oleifera leaves (common name: drum stick tree; family: Moringaceae; Anwar et al. 2007) and Embelia ribes seeds (common name: false black pepper; family: Myrsinaceae; Kekuda et al. 2009; Swarnkar et al. 2009) were selected in this study to assess their anthelmintic efficacy against H. contortus. This communication describes the anthelmintic efficacy of the ethanolic and aqueous extracts of Moringa oleifera and Embelia ribes against different life-cycle stages of H. contortus.
Materials and methods
Selection of plant materials and preparation of extracts
Based on literature search for the anthelmintic property-bearing plants which are abundantly and easily found in India, Moringa oleifera (common name: Drum stick tree, Family: Moringaceae) leaves (Anwar et al. Reference Anwar, Latif, Ashraf and Gilani2007) and Embelia ribes (Common name: False black pepper, Family: Myrsinaceae) seeds (Kekuda et al. Reference Kekuda, Kumar, Nishanth and Sandeep2009; Swarnkar et al. Reference Swarnkar, Singh, Khan and Bhagwan2009) were selected for assessment of their anthelmintic efficacy against H. contortus. Fresh leaves of M. oleifera were collected from the premises of Indian Veterinary Research Institute, Bareilly, India, and Embelia seeds were procured from the local market. Moringa leaves were shade dried till complete removal of moisture and macerated to powder form. A total of 480 grams of powder was used for extraction using 70.0% ethanol (6 litres). The crude extract obtained was filtered through Whatman filter paper (No. 3) and then filtrate was concentrated using rotary evaporator. Semisolid extract obtained in the process was weighed and percent yield was calculated. Subsequently, stock solution (100 mg/mL) was prepared in 1.0% dimethyl sulphoxide (DMSO), labelled as M. oleifera ethanolic extract (MOEE), and stored till further use. In a similar manner, E. ribes ethanolic extract (EREE) was also prepared.
Moringa aqueous extract (4.0%) was freshly prepared by adding 4.0 g powder to 100 mL distilled water and by heating the same at 60°C for 45 min with thorough mixing (Amrulloh et al. Reference Amrulloh, Fatiqin, Simanjuntak, Afriyani and Annissa2021). This mixture was cooled down to room temperature and then filtered using Whatman filter paper (No.3). Filtrate obtained was designated as M. oleifera aqueous extract (MOAE). Likewise, 4.0% Embelia ribes aqueous extract (ERAE) was also prepared from the macerated seeds. All the plant extracts were stored in airtight vials at −20°C till further use to avoid fungal growth.
Assessment of plant secondary metabolites
The aqueous and ethanolic extracts of Moringa oleifera and Embelia ribes were analysed for their phytochemical constituents. The concentrations of total phenolics, tannins, alkaloids, and flavonoids were determined following established methods described by Waterhouse (2002), Scalbert (1992), John et al. (Reference John, Sulaiman, George and Reddy2014), and Lydia et al. (2016), respectively, with the slight modifications.
Anthelmintic efficacy assessment
Harvesting of strongyle eggs, coproculture, and recovery of adult H. contortus
Fresh faecal samples were collected from the rectum of sheep housed at the Livestock Farm Complex of the Institute and brought to laboratory for analysis. Faecal samples positive for strongyle ova were pooled and subjected to concentration of the strongyle egg using saturated salt solution (Soulsby Reference Soulsby1982). Concentrated eggs were washed with distilled water and finally half of the eggs were suspended in phosphate-buffered saline (PBS, pH: 7.2) to obtain concentration of about 100–150 eggs/100 μL PBS. Remaining eggs were cultured for the development of larvae (L3 stage) for further identification. Adult H. contortus were recovered from the sheep abomasa procured from the local slaughter house. The freshly harvested eggs and adult worms were subsequently employed in different in vitro techniques.
In vitro techniques
Different concentrations of the ethanolic extracts viz., 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 mg/mL, were prepared from the stock solution (100 mg/mL), whereas aqueous extracts were used as such without further dilution. Albendazole (1.0 mg/mL), PBS, and DMSO (1.0%) were also used as positive, negative, and solvent controls, respectively. The final concentration of 100–150 eggs/100 μL PBS was used for each assay. All tests were performed in duplicates.
Egg hatch assay (EHA)
The methodology described by Costa et al. (Reference Costa, Bevilaqua, Camurça-Vasconcelos, Maciel, Morais, Castro, Braga and Oliveira2008) was followed for the EHA incorporating minor adjustments as necessary. Briefly, freshly diluted eggs (100 μL) were added to each well of the 24-welled cell culture plate, followed by addition of 400 μL of PBS to make the final volume to 500 μL. Equal volume (500 μL) of different concentrations of herbal extracts, positive drug (albendazole) control, DMSO, etc., was added to their respective wells. The plate was incubated at 27°C for 48 hours with 80% relative humidity (RH). Egg hatching was terminated by adding 500 μL of 10% formalin. Total number of eggs and hatched larvae was counted under microscope (100×).
Larval development assay (LDA)
LDA was conducted in accordance with the procedures outlined by Tadesse et al. (Reference Tadesse, Eguale, Giday and Mussa2009) with slight modifications. Briefly, 100 μL of the diluted eggs were dispensed into individual wells of a 24-well cell culture plate. Each well was supplemented with 20 μL of larval nutritive medium (composed of 3 mL Earle’s salt solution mixed with 27 mL of yeast extract) and 20 μL of Amphotericin B (0.3 mg/mL) to prevent fungal growth. The final volume was adjusted to 500 μL by adding PBS. These plates were then placed in an incubator at 27°C for 24 hours to allow the first-stage larva to develop from the eggs. Subsequently, equal volumes (500 μL) of plant extracts, positive, negative, and solvent controls were added and all plates were further incubated at 27°C with 80% RH for an additional 6 days. Larval development was halted by the addition of 500 μL of 10% formalin. The number of unhatched eggs and developed larvae (L1, L2, L3 stages) was enumerated using a compound microscope (100× and 400× magnifications).
Adult mortality assay (AMA)
AMA was performed based on the method described by Lal et al. (Reference Lal, Chandra, Raviprakash and Sabir1976), with minor adjustments. Actively motile adult H. contortus were carefully picked up from the sheep abomasa and subsequently washed with PBS. Ten worms per well were transferred to the Petri plate containing different plant extracts in duplicate wells. Positive (Albendazole @ 1.0 mg/mL; Levamisole @ 1.0 μL/mL) as well as a negative control (PBS) were also included. The plates were then placed in an incubator at 37°C, and the motility of the worms was observed at intervals of 0, 1, 3, 6, 8, 10, and 12 hours post-incubation. The total number of motile (live) and immotile (dead) worms was counted using a stereo microscope. Following the in vitro assays, the herbal product with promising ovicidal, larvicidal, and adulticidal effects was further selected for in vivo trial.
In vivo trial
The faecal egg count reduction test (FECRT) was performed as per the procedure mentioned by Coles et al. (Reference Coles, Jackson, Pomroy, Prichard, von Samson-Himmelstjerna, Silvestre, Taylor and Vercruysse2006). Briefly, a total of 40 naturally infected sheep (3–6 months of age, >500 eggs per gram) were randomly distributed in four groups (10 animals per group). Group I animals received MOAE (15.0%) @ 1.0 mL/kg body weight (BW) orally on alternate days till day 9 post-treatment (i.e. total 5 drenchings). Animals of Groups II and III were treated orally with fenbendazole @ 5.0 mg/kg BW and levamisole @ 7.5 mg/kg BW, respectively; whereas animals of Group IV did not receive any kind of treatment and acted as control group. Faecal samples were collected from animals of Groups I, II, and IV on day 0 and day 10 post-treatment, whereas same was collected on day 7 post-treatment from levamisole-treated (Group III) animals. All samples were screened by the modified McMaster egg counting technique. Pre- and post-treatment EPG was recorded for each animal involved in the experiment. Faecal cultures were prepared during each sampling from pooled faecal samples of each treatment group in order to allow the development of third-stage larvae and identification of the genus of the GI nematodes. The cultured larvae from each group were examined under microscope and were morphologically identified as H. contortus larvae. Later, genomic DNA was extracted from every single larva. Allele-specific PCR (AS-PCR) was performed to know the resistance status against benzimidazoles (Coles et al. Reference Coles, Jackson, Pomroy, Prichard, von Samson-Himmelstjerna, Silvestre, Taylor and Vercruysse2006). The procedure described by Barrère et al. (Reference Barrère, Beech, Charvet and Prichard2014) targeting the Hco-acr-8b gene was followed to know the resistance status against levamisole in surviving larvae of H. contortus.
Statistical analyses
Statistical significance of the effectiveness of the plant extracts in different in vitro tests was assessed using one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) test in SPSS version 21.0 software. The inhibition concentration 50 (IC50) value was determined for MOEE and EREE through nonlinear regression analysis (curve fit) in GraphPad Prism 5. The result of in vivo trial was subjected to paired t-test to analyse the significant differences between mean EPG outcomes of different groups (I to IV) pre- and post-treatment.
Results
The percent yield of the crude ethanolic extracts of M. oleifera (leaves) and E. ribes (seeds) was found to be 21.3% and 5%, respectively. The ethanolic extracts contained various phytoconstituents, including phenolic compounds, tannins, and alkaloids. In contrast, the powdered forms of Moringa and Embelia contained the same constituents, except for alkaloids (Table 1). The coproculture and larval identification revealed that Haemonchus contortus constituted 90% of the recovered strongyle larvae, with the remaining proportion comprising other gastrointestinal nematodes. Therefore, the assay results predominantly reflect the activity of the extracts against H. contortus. In the EHA, highest inhibition of the egg hatching process was exhibited by MOAE, i.e. 89.12% followed by MOEE (87.77% @ 16 mg/mL). However, the ANOVA analysis did not detect any significant difference between MOAE (4.0%) and MOEE @ 16.0 and 32.0 mg/mL concentrations. Least inhibition was observed with ERAE (12.83%). The standard drug albendazole, used in the study as positive control, could only inhibit 50.81% of egg hatching (Table 2). In the LDA, with increasing conc. of the ethanolic extracts (MOEE, EREE), the proportions of unhatched eggs and L1 larvae also increased gradually. Aqueous extract of Moringa and Embelia caused 89.94% and 80.40% larval inhibition. The differences in the mean inhibition % of the ethanolic (@ 8.0, 16.0, 32.0 mg/mL) and aqueous Moringa extract were not statistically significant in LDA. Likewise, Embelia ethanolic (@ 8.0, 16.0 mg/mL) and aqueous extract also did not show any significant difference in their efficacy to impair larval development in LDA. Albendazole used in the trial showed 79.69% inhibitory effect. The effect of the ethanolic extracts of Moringa and Embelia in EHA as well as LDA was concentration-dependent with minimal deviations (Fig. 1, Table 2). The IC50 values for MOEE (EHA, LDA) and EREE (LDA) are 5.721, 4.131, and 2.882, respectively (Fig. 1). However, the EREE failed to produce 50% inhibition of egg hatch process in EHA at all the tested concentrations. In the adult mortality assay (AMA), about 50% mortality was observed 8 hours post-incubation with the MOEE and EREE (4.0, 8.0, 16.0 mg/mL conc.). In case of aqueous extracts, 50.0% mortality occurred at 6 hours post-incubation. Early mortality (1 hour post-incubation) of worms was appreciated first in the albendazole-supplemented Petri plates (about 50% mortality). The rate of worm mortality in MOAE was more in comparison to MOEE at any given point of time. Complete mortality was achieved at 12 hours post-incubation with the aqueous and ethanolic extracts (8.0 mg/mL and 16.0 mg/mL) of Moringa. In contrary to this, ERAE and EREE caused only 50% and 80% worm mortality, respectively, at 12 hours post-incubation. Moringa aqueous extract was found to be more effective in all the in vitro assays and hence chosen for in vivo trial.
Phytoconstituents detected in Moringa oleifera leaves and Embelia ribes seeds
Table 1. Long description
The table consists of five columns and five rows including the header.
Column headers from left to right are: Plant extracts, Phenolic compounds (mu g/g), Tannins, Alkaloids (mu g/g), and Flavonoids (mu g/g).
Row 1: Moringa oleifera (M O E E) contains 693.56 Phenolic compounds, 6.23 percent Tannins, 167.39 Alkaloids, and 2292.84 Flavonoids.
Row 2: M. oleifera (leaf powder) contains 250.86 Phenolic compounds, 3.11 percent Tannins, no Alkaloids detected, and 362.32 Flavonoids.
Row 3: Embelia ribes (E R E E) contains 853.42 Phenolic compounds, 7.79 percent Tannins, 168.36 Alkaloids, and 4298.75 Flavonoids.
Row 4: E. ribes (seed powder) contains 123.53 Phenolic compounds, 3.11 percent Tannins, no Alkaloids detected, and 203.46 Flavonoids.
Mean inhibition percentage of different extracts of Moringa oleifera and Embelia ribes in egg hatch assay (EHA) and larval development assay (LDA)
Table 2. Long description
The table consists of five columns. The first column lists the treatment type and concentration. Columns two and three show results for Moringa oleifera leaves in E H A and L D A respectively. Columns four and five show results for Embelia ribes seeds in E H A and L D A respectively.
Ethanolic extract concentrations and results:
* 1 mg/mL: Moringa E H A 5.51 plus or minus 0.51, L D A 31.44 plus or minus 3.42; Embelia E H A 10.56 plus or minus 0.21, L D A 32.30 plus or minus 2.70.
* 2 mg/mL: Moringa E H A 8.45 plus or minus 0.83, L D A 27.26 plus or minus 3.01; Embelia E H A 11.91 plus or minus 1.87, L D A 32.95 plus or minus 0.39.
* 4 mg/mL: Moringa E H A 15.78 plus or minus 2.69, L D A 53.99 plus or minus 2.80; Embelia E H A 14.37 plus or minus 1.50, L D A 70.21 plus or minus 0.21.
* 8 mg/mL: Moringa E H A 74.98 plus or minus 2.20, L D A 79.83 plus or minus 1.85; Embelia E H A 19.21 plus or minus 1.28, L D A 76.44 plus or minus 0.12.
* 16 mg/mL: Moringa E H A 87.77 plus or minus 1.42, L D A 80.58 plus or minus 2.18; Embelia E H A 22.69 plus or minus 1.13, L D A 77.90 plus or minus 3.35.
* 32 mg/mL: Moringa E H A 82.20 plus or minus 1.51, L D A 86.97 plus or minus 1.56; Embelia E H A 22.66 plus or minus 2.09, L D A 59.22 plus or minus 2.07.
Other treatments and controls:
* Aqueous extract 4.0 percent: Moringa E H A 89.12 plus or minus 1.44, L D A 89.94 plus or minus 2.63; Embelia E H A 12.83 plus or minus 2.10, L D A 80.40 plus or minus 3.21.
* Albendazole 1.0 mg/mL: Moringa E H A 50.81 plus or minus 2.63, L D A 79.69 plus or minus 2.03; Embelia E H A 40.67 plus or minus 1.88, L D A 77.38 plus or minus 2.01.
* P B S pH 7.2: Moringa E H A 3.81 plus or minus 0.54, L D A 29.02 plus or minus 4.15; Embelia E H A 5.72 plus or minus 0.75, L D A 35.383 plus or minus 0.9002.
* D M S O 1 percent: Moringa E H A 11.77 plus or minus 1.80, L D A 20.08 plus or minus 0.93; Embelia E H A 4.55 plus or minus 0.69, L D A 24.202 plus or minus 1.0016.
Data are presented as mean ± SD. Different superscript letters (a-f) within a column indicate significant differences (p < 0.05) according to Tukey’s HSD test.
Dose-response relationship and the IC50 of M. oleifera ethanolic extract (MOEE) and E. ribes ethanolic extract (EREE) against H. contortus eggs and larvae in egg hatch assay (EHA) and larval development assay (LDA). A slight discrepancy between the intersecting IC₅₀ points on the graph and the mathematically calculated IC50 values may arise because the x-axis (concentration) is plotted on a logarithmic scale, and visually determining the IC₅₀ can occasionally result in minor inconsistencies. The IC50 for EREE in EHA is >32 mg/mL because EREE did not cause 50% inhibition of the egg hatching process at the tested concentrations (1–32 mg/mL). The IC50 values are presented with their corresponding 95% confidence intervals. Specifically, the IC50 values (95% CI) were 5.721 mg/mL (4.436–7.379) for MOEE-EHA, 4.131 mg/mL (3.037–5.619) for MOEE-LDA, 5.652 mg/mL (3.933–8.124) for EREE-EHA, and 2.882 mg/mL (0.020–408.1) for EREE-LDA.
Figure 1. Long description
A line graph with a logarithmic x-axis labeled Concentrations in mg per mL ranging from 1 to 32 and a y-axis labeled Inhibition percentage ranging from 0 to 100. A horizontal dashed line marks the 50 percent inhibition threshold. Four data series are plotted with error bars.
* E R E E, L D A (green inverted triangles) shows the highest initial inhibition, rising sharply from 1 to 4 mg per mL where it crosses the 50 percent line, peaking near 80 percent at 16 mg per mL, and dropping to 60 percent at 32 mg per mL. I C sub 5 0 is 2.882 mg per mL.
* M O E E, L D A (orange squares) starts at 30 percent, crosses the 50 percent line at 4 mg per mL, and increases steadily to nearly 90 percent at 32 mg per mL. I C sub 5 0 is 4.131 mg per mL.
* M O E E, E H A (pink circles) starts low at 5 percent, rises sharply between 4 and 8 mg per mL, crossing the 50 percent line just after 4 mg per mL. I C sub 5 0 is 5.721 mg per mL.
* E R E E, E H A (yellow triangles) shows the lowest efficacy, remaining below 25 percent inhibition across all tested concentrations. I C sub 5 0 is greater than 32 mg per mL.
Vertical dashed lines indicate the specific concentration points where the L D A and E H A curves intersect the 50 percent inhibition mark.
During the in vivo trial, faecal samples were collected on day 0, 7, and 10 post-treatment and processed for calculation of EPG by modified McMaster technique. The sheep used in the in vivo trial were naturally infected with a predominantly H. contortus population. Pre-treatment coproculture showed that H. contortus represented around 90% of the recovered larvae, with a minor proportions of other strongyle genera. The FEC reduction values in MOAE (Group I), fenbendazole (Group II), and levamisole (Group III) treated groups were determined as 72.42, 70.92, and 89.01%, respectively (Table 3). The paired t-test conducted for each treatment group exhibited statistically significant (p <0.05) reduction in EPG pre- and post-treatment, and as anticipated there was no difference in the EPG values of the animals of the control/untreated group (Group IV) at the end of the trial. Third-stage larvae (L3) recovered from coproculture of pooled faeces in all three treatment groups were microscopically identified as those of H. contortus. Aqueous extract of Moringa was effective with the five alternate dose regimen @ 150 mg/kg BW. The FECR% values indicated presence of fenbendazole- and levamisole-resistant worms in the animals. Additionally, in AS-PCR, amplification of 222 bp (resistant allele specific) size products indicated benzimidazole resistance in the treatment-surviving larvae. Amplification of the Hco-acr-8b gene (256 bp) indicated levamisole resistance. Our study findings from both FECR test and molecular analysis confirmed presence of benzimidazole- and levamisole-resistant H. contortus worms in the sheep of Livestock Farm Complex, IVRI, Izatnagar.
Mean faecal egg count and FECR (%) in sheep treated with M. oleifera aqueous extract and selected anthelmintics
Table 3. Long description
The table consists of five columns: Animal groups, Mean E P G plus or minus S E for 0th day, 7th day, and 10th day, and F E C R percentage.
* Group-I (M O A E at 150 mg/kg B W): 0th day mean is 9790.0 plus or minus 1767.13. 10th day mean is 2700 plus or minus 975.5 with an asterisk. F E C R is 72.42 percent.
* Group-II (Fenbendazole at 5 mg/kg B W): 0th day mean is 12310.0 plus or minus 4411.81. 10th day mean is 3580.0 plus or minus 1295.44 with an asterisk. F E C R is 70.92 percent.
* Group-III (Levamisole at 7.5 mg/kg B W): 0th day mean is 9550.00 plus or minus 2178.34. 7th day mean is 1050.00 plus or minus 246.86 with an asterisk. F E C R is 89.01 percent.
* Group-IV (Untreated control): 0th day mean is 3340.00 plus or minus 578.54. 10th day mean is 3470.00 plus or minus 533.96.
The asterisk indicates significant differences (p < 0.05) between group means pre- and post-treatment based on paired t-test statistics.
The asterisk (*) indicates significant differences (p <0.05) between group means pre- and post-treatment as concluded by paired t-test statistics.
Discussion
Indiscriminate use of the available anthelmintics to control GIN infection in general and H. contortus in particular has led to development of resistant strains. Effective management of these resistant nematodes requires alternative interventions such as use of effective vaccines, biological control agents, and plant-based dewormers. Identification of potential plants with desired level of efficacy may help in formulations of an integrated and sustainable control strategy for GI nematodes of small ruminants. The plant secondary metabolites (tannins, saponins, flavonoids, plant cysteine proteases, alkaloids, polyphenolic compounds, terpenes, etc.) are the main bioactive compounds that show anthelmintic activity (Ayers et al. Reference Ayers, Zink, Mohn, Powell, Brown, Murphy, Brand, Pretorius, Stevenson, Thompson and Singh2007; Zhu et al. Reference Zhu, Dai, Yang and Qiu2013; Oliveira et al. Reference Oliveira, Costa, Lima, Silva, Ribeiro, Mesquista, Rocha, Tangerina and Vilegas2017; Spiegler et al. Reference Spiegler, Liebau and Hensel2017) and are present in different proportions in various plant species, and thus their anti-parasitic effect also varies to a great extent. Identification of potential plants with desired level of efficacy may help in formulations of an integrated and sustainable control strategy for GI nematodes of small ruminants. In the present study anthelmintic activity of M. oleifera (leaves) and E. ribes (seeds) was explored.
Different plant extracts like MOEE, MOAE, ERAE showed a considerable level of inhibitory effects on the egg hatching, larval development, and adult worm mortality. The ethanolic and aqueous extracts of Moringa oleifera exhibited strong ovicidal and larvicidal activities, with the aqueous extract showing efficacy comparable to or greater than the ethanolic extract, indicating that water-soluble phytoconstituents also contribute significantly to its anthelmintic activity. In contrast, the ethanolic extract of Embelia ribes showed limited ovicidal activity but pronounced inhibition of larval development, suggesting stage-specific susceptibility of the parasite. The Moringa aqueous extract showed almost 90% inhibitory effect on egg hatching, larval development, and adult worm mortality, whereas the Embelia aqueous extract showed only 80% larvicidal activity. According to Cabardo and Portugaliza (Reference Cabardo and Portugaliza2017), the in vitro ovicidal effect of ethanolic and aqueous extract of M. oleifera seeds against H. contortus was 95.89% and 81.72% at 15.6 mg/mL conc. In the larvicidal assay, the ethanolic and aqueous extracts exhibited 56.94% and 92.50% efficacy at 7.8 mg/mL, respectively. It appears that plant extracts prepared from Moringa seeds as well as leaves produce good in vitro ovicidal and larvicidal effects. The anthelmintic efficacy of Moringa leaf extracts (ethanolic, infused aqueous, and macerated aqueous extract) was evaluated by Tayo et al. (Reference Tayo, Poné, Komtangi, Yondo, Ngangout and Mbida2014) at different conc. (0.625, 1.25, 2.5, 3.75, and 5 mg/mL) against different stages of H. contortus (fresh eggs, embryonated eggs, L1, L2). They inferred that the ethanolic extract had highest larvicidal effect, infused aqueous extract exhibited more effects on eggs, and the macerated aqueous extract was effective in inhibiting egg embryonation, egg hatching, and caused larval mortality. The results of the present study are in line with the reports of Tayo et al. (Reference Tayo, Poné, Komtangi, Yondo, Ngangout and Mbida2014). The differences observed between the ethanolic and aqueous extracts could be due to the degree of solubility of the phytoconstituents in different solvents (Saini et al. Reference Saini, Sivanesan and Keum2016). Additionally, the effect of the Moringa extracts has also been reported against other organisms, viz., Pheritima posthuma (Kekuda et al. Reference Kekuda, Kumar, Nishanth and Sandeep2009), Tubiflex tubiflex (Anitha and Kalaivani Reference Anitha and Kalaivani2018), Eimeria papillata oocyst (Abdel-Tawab et al. Reference Abdel-Tawab, Abdel-Haleem, Abdel-Baki, Al-Quraishy and El-Mallah2020), and Fasciola gigantica (Hegazi et al. Reference Hegazi, Megeed, Hassan, Abdelaziz, Toaleb, El Shanawany and Aboelsoued2018).
The ethanolic extract of Embelia didn’t show much effect on inhibiting egg hatching, but moderate effects on larval development and adult mortality were recorded. In contrast, the aqueous extract of Embelia exhibited a strong larvicidal effect but showed weak ovicidal and adulticidal effects. However, according to Swarnkar et al. (Reference Swarnkar, Singh, Khan and Bhagwan2009), both aqueous and alcoholic extracts of Embelia seeds showed potent ovicidal and mild larvicidal activity. As per their report, aqueous Embelia extract is comparatively better than its counter alcoholic, ether, and chloroform extracts. The ethanolic extracts of Moringa and Embelia used in this study exhibited moderate IC50 values both in EHA and LDA, indicating that with standardised dose rate and in vivo trials, the extracts could act more effectively against H. contortus.
Based on the overall performance of both plant extracts in the in vitro testing, MOAE (five alternate doses @ 150 mg/kg BW) was further selected for in vivo studies. The FECRT results revealed 72.42% overall EPG reduction in the MOAE-supplemented animals. Pedraza-Hernández et al. (Reference Pedraza-Hernández, Elghandour, Khusro, Salem, Camacho-Diaz, Barbabosa-Pliego and Salem2021) conducted an in vivo study using hydro-alcoholic (water:ethanol:methanol – 80:10:10) extract (@ 0, 30, 60 mL/goat in each treatment group) of Moringa leaves. They reported significant anthelmintic activity of the extract (@ 60 mL/goat) against Trichuris and Ostertagia spp. and also explored its positive effect in improvising the daily weight gain and feed conversion ratio in the animals. There is a paucity of literature on in vivo trials for assessment of the anthelmintic activity of Moringa leaf extracts against H. contortus. Many in vivo studies have been conducted on Embelia fruit and seed extracts. Swarnkar et al. (Reference Swarnkar, Singh, Khan and Bhagwan2009) reported a reduction of 46.3% and 65.6% in FEC after using aqueous and alcoholic extracts of Embelia seeds, respectively, on day 10 post-treatment. The mixture of the powdered forms of E. ribes fruits and Vernonia anthelmintica seeds in 1:1 ratio when given @ 600 mg/kg BW orally to the animals in the treatment group resulted in 62.07% reduction in faecal egg count (Sinha et al. Reference Sinha, Singh, Pal, Maurya, Singh and Shekhar2021). Javed and Akhtar (Reference Javed and Akhtar1990) used the crude powder of these two plants (1:1 ratio), their equivalent water and methanol extract each @ 2 g/kg BW, and reported FECR % of 83, 27, and 93%, respectively. The active component Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone) present in E. ribes, alone or in combination with other phytochemicals, is assumed to exert anthelmintic effect.
The FECRT data are key component for determining the efficacy of the anthelmintics against GINs where the average FECRT value for all trials submitted must be ≥90%. In our study, the FECR test revealed significant result (72.42%) in the Moringa aqueous extract supplemented group which could further be improved by making alterations in the dose regimen. The FECRT values for both albendazole and levamisole treated group was less than 90% (Kaplan et al. Reference Kaplan, Denwood, Nielsen, Thamsborg, Torgerson, Gilleard, Dobson, Vercruysse and Levecke2023), indicating development of moderate level of resistance among the worm populations against these two anthelmintics.
The current study demonstrated reasonably good efficacy (in vitro) of the ethanolic and aqueous extract of M. oleifera leaves against all three stages of H. contortus. However, ethanolic and aqueous extracts of the E. ribes showed good larvicidal effect. Further in vivo trial employing aqueous extract of Moringa leaves showed promising anthelmintic efficacy against gastrointestinal nematodes infecting sheep. However, further in vivo investigations are needed to optimise the anthelmintic efficacy of these extracts. This could involve exploring potential synergies among herbal (Moringa and Embelia) extracts at varying ratios, standardising doses, combining chemical anthelmintics with different proportions of herbal extracts, etc. Such a multifaceted approach holds promise for effectively combating gastrointestinal nematodes. Nonetheless, additional studies are essential to ascertain the most practical and effective dosage of the extract for use in parasitised animals.
Acknowledgements
The authors are thankful to the Director, ICAR-IVRI, Izatnagar, for providing the necessary facilities for completing this research work.
Author contribution
Hira Ram: Study conception and design; Shamli: Performed research; Shamli, Rasmita Panda, A. Nandini, Devendra Prasad Pateer: Data analysis and formal writing; Hira Ram, Rajat Garg, Narayan Dutta: Review and editing.
Competing interest
The authors declare that they do not have any conflicts of interest.
Statement of animal rights
This study was conducted in strict accordance with ethical guidelines for the care and use of animals in research. All experimental protocols involving animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC).
Consent for publication
Not applicable.
