Anthelminthic Activity of Moringa oleifera Leaf Extracts Evaluated in Vitro on Four Developmental Stages of Haemonchus contortus from Goats

Haemonchus contortus is a blood-sucking abomasal helminth of small ruminants responsible for major economic losses to small farmers worldwide. Widespread resistance to synthetic anthelminthics has stimulated a need for alternative strategies of parasite control, among which is the use of medicinal plants with natural anthelminthic properties. This study assessed in vitro the efficacy of infused and macerated aqueous extract as well ethanolic extract of Moringa oleifera against fresh eggs, embryonated eggs, L1 and L2 larvae of H. contortus. For this purpose, five different concentrations (0.625, 1.25, 2.5, 3.75 and 5 mg/ml) were prepared from dry extracts via serial dilutions with distilled water. Fresh eggs obtained from artificially infected goat feces were exposed to these different concentrations for 48 hours, while embryonated eggs and larvae were exposed for 6 and 24 hours respectively. Distilled water and 1.5% DMSO were used as negative control. The results were expressed in terms of mean inhibition percentage of egg embryonation, mean inhibition percentage of egg hatch and mean percentage of larval mortality. An overview of results revealed that ethanolic leaf extract of M. oleifera was most efficient on eggs by inhibiting 60.3% ± 8.2% and 92.8% ± 6.2% eggs embryonation at 3.75 and 5 mg/ml respectively with a significant difference (P ˂ 0.05), which contributed to obtaining the lowest LC50 value of 0.985 mg/ml. This extract also inhibited 99% ± 2% egg hatching of H. contortus at 5 mg/ml with an LC50 value of 1.7 mg/ml. Concerning activity on larvae, the ethanolic extract was also most potent against them by inducing 98.8% ± 2.5% and 100% ± 0% mortality of L1 and L2 larvae at 5 mg/ml respectively. Infused aqueous extract was more efficient on eggs than on larvae with an IC50 value less than 2 mg/ml and an LC50 value more than 3.5 mg/ml. Macerated aqueous extract showed good activity Corresponding author. G. Mbogning Tayo et al. 1703 against the four developmental stages with LC50 values ranging from 2.08 mg/ml for L2 larvae to 2.92 mg/ml for L1 larvae and 2.37 to 2.52 mg/ml for embryonated and fresh eggs respectively. The current study showed that all three extracts of M. oleifera tested possessed potential ovicidal and larvicidal activities against H. contortus. However, further in vivo studies are necessary to validate the anthelminthic property of this plant.


Introduction
Livestock is an important prospective sector which may contribute in solving problems of small farmers and by such, help in poverty alleviation [1].Small ruminants particularly goats have been considered the most important aspect of livestock throughout the world.In 2010, world caprine livestock counted 920 million animals, with more than 90% in Asia and Africa [2].In Cameroon, Labonne et al. [3] registered 7 million goats, with half of this population concentrated in the Extreme-North Region.Parasitism by gastro-intestinal nematodes (GINs) is a major constraint in the production of goats in tropical countries.They can affect production through weight loss, diarrhea, anemia, reduction in milk and wool production, reproduction changes as well as mortality in the case of heavy infectations [4].Actually, a majority of nematodes affecting the intestinal tract of small ruminants belong to the Order Strongylidae, with principal genera being Haemonchus, Teladorsagia, Cooperia, Trichostrongylus, Nematodirus, Chabertia and OEsophagostomun [5].Compared to other nematodes, H. contortus is by far a highly pathogenic parasite of small ruminants, capable of causing acute disease and high mortality in all classes of livestock [6].Up to now, a huge amount of money is spent annually worldwide to combat helminth parasites in livestock, the principal mode of control being based on the repeated use of commercial anthelminthic drugs such as levamisol, morantel, thiabendazole, mebendazole, albendazole, ivermectin and dormactin.However, nematodes have developed resistance against several families of anthelminthics.Moreover, toxicity due to inappropriate dose administration and risk of drug residues in animal products are other big problems associated with the use of synthetic drugs [7].In most developing countries like Cameroon, small holder farmers have limited access to such drugs and veterinary services due to either high cost or unavailability.Therefore, most of the farmers rely on ethno-veterinary treatment.Medicinal plants are considered as an alternative source of compounds that are biodegradable into non-toxic products and sustainable methods readily adaptable to rural farming communities.Moringa oleifera is considered as one of the most useful trees as almost every part can be used for food or has some other beneficial properties.In the tropics, it is used as forage for livestock and in many countries; it is used to treat various ailments [8].In Cameroon, M. oleifera is used to treat asthma, anemia, intestinal worms, cardio-vascular disorder, headache, skin infections and others (personal communication).Extracts from this plant have several pharmacological effects such as anthelminthic, anti-inflammatory, antimicrobial, anti-oxydant, hepato-protective, anti-glycemic and anti-dislipidemia [8]- [13].As far as literature on this plant is concerned, there is no published work on in vitro anthelminthic activity of Moringa oleifera against Haemonchus contortus.The aim of the present study was thus to investigate the in vitro activities of aqueous and ethanolic leaf extracts of M. oleifera on four free-living stages of H. contortus from goats.

Collection and Storage of Plant Material
Leaves from mature trees were collected at the teaching and research farm of the University of Dschang-Cameroon.A branch of leaves was taken to the National Herbarium of Cameroon where it was identified under the reference number 42885/HNC as leaves of Moringa oleifera Lam.The collected plant material was dried in shade, at ambient temperature for three weeks.Dried leaves were ground to powder and stored in airtight plastic bags in the Laboratory of Biology and Applied Ecology of the University of Dschang.

Plant Extracts Preparation
The infused and macerated aqueous extracts as well as ethanolic extract were prepared to compare their activities.Extraction was done according to the procedure described by Wabo Pone et al. [14] [15], at the end of which we obtained different dried extracts.Each dried extract was used to prepare a stock solution which was then diluted with distilled water to obtain five different solutions of concentrations 1.25, 2.5, 5, 7.5 and 10 mg/ml.The final tested concentrations were 0.625, 1.25, 2.5, 3.75 and 5 mg/ml.

Parasites Donor Goat
Goats' abomasums were obtained from the abattoir of the "Marché B" of Dschang town after necropsy of animals.Adult female Haemonchus contortus were recovered from abomasums.These female worms were crushed to liberate eggs.The eggs were then cultured in vitro in Petri dishes at room temperature for seven days.At the end of the 7 th day, infective larvae were harvested.About 2500 larvae were inoculated into a worm-free goat kept indoors in a separate house at the teaching and research farm of the University of Dschang throughout the study period.This goat served as H. contortus egg donor for subsequent in vitro trials.

Recovery of Nematode Eggs
Feces directly collected from the rectum of the donor goat mentioned above were used in recovering eggs according to the procedure carried out by Wabo Poné et al. [15].

Recovery of Nematode Larvae
L 1 and L 2 larvae were obtained from eggs recovered above according to Mbogning Tayo et al. [16].

Evaluation of Ovicidal and Larvicidal Activities
Egg embryonation assay using fresh eggs, egg hatch assay using embryonated eggs and larval mortality assay using L 1 and L 2 larvae were performed according to Wabo Poné et al. [17] [18], to evaluate the ovicidal and larvicidal efficacy of M. oleifera leaf extracts.Each test was repeated four times for each extract and control (distilled water and 1.5% DMSO).

Statistical Analysis
Comparison of the mean inhibition percentage of egg embryonation, mean inhibition percentage of egg hatch and mean percentage of larval mortality at different concentrations with control was performed by one-way analysis of variance (ANOVA).Statistical analysis was performed by using the software SPSS version 17.0.The post hoc statistical significance test employed was Duncan, differences between the means were considered significant at P < 0.05.The 50% inhibitory concentration (IC 50 ) and lethal concentration (LC 50 ), i.e., effective concentration to inhibit 50% of the eggs and to kill 50% of larvae were determined using the regression lines of the probit according to decimal logarithm of the concentrations.1.From this table, we observed that negative controls (distilled water and 1.5% distilled water) had no effect on egg embryonation while M. oleifera extracts inhibited embryonation in a concentration dependent fashion.Ethanolic extract was efficient at 0.625 mg/ml by inhibiting 51.7% ± 20.7% egg embryonnation, reaching 92.8% ± 6.2% at 5 mg/ml with a significant difference (P < 0.05).Infused and macerated aqueous extracts also inhibited embryonation with mean efficacy of 69% ± 5.8% and 94.5% ± 4% at 5 mg/ml respectively.The IC 50 s calculated from equations of regression lines of probit according to the decimal logarithm of concentrations (Figure 1) were 2.52, 1.52 and 0.985 mg/ml for macerated, infused aqueous extract and ethanolic extract respectively.

Efficacy of M. oleifera leaf extracts in inhibiting egg embryonation of Haemonchus contortus at different concentrations is presented in Table
Table 2 shows activity of extracts in inhibiting egg hatch of H. contortus at different concentrations.Like on embryonation, distilled water and 1.5% DMSO did not affect egg hatch while extracts presented a concentration   dependent activity.Macerated aqueous extract and ethanol extract inhibited more than 60% egg hatch at 2.5 mg/ml, reaching 90.2% ± 8.4% and 99% ± 2% at 5 mg/ml respectively.The infused aqueous extract inhibited 69.2% ± 17.1% and 97.9% ± 4.2% egg hatch at 3.75 and 5 mg/ml respectively with a significant difference (P < 0.05).IC 50 s values of 2.37, 1.75 and 1.74 mg/ml were obtained for macerated, infused and ethanolic extracts respectively from equations of regression lines of the probit of egg hatch inhibition according to decimal logarithm of concentrations (Figure 2).
Concerning larvicidal activity, Table 3 and Table 4 present the efficacy of M. oleifera extracts in inducing L 1 and L 2 larva mortality at different respectively.From Table 3, infused aqueous extract showed weak activity on L 1 larvae, inducing only 50.5% ± 8.8% mortality at 5 mg/ml.However, macerated aqueous extract induced 89.6% ± 8.8% at 5 mg/ml while at the same concentration ethanolic extract registered 98.8% ± 2.5% L 1 larva mortality.LC 50 s values of L 1 larvae calculated from equations of regression lines (illustrated on Figure 3) were 7.83, 2.92 and 1.89 mg/ml for infused, macerated aqueous extracts and ethanolic extract respectively.As seen from Table 4 the ethanolic extract was most efficient in inducing 98.8% ± 3.1% and 100% ± 0% L 2 larva mortality at 3.75 and 5 mg/ml respectively, with no significant difference (P ˃ 0.05).At these same concentrations, aqueous extracts also induced L 2 mortality even though the mean mortality rates were lower than those obtained with ethanolic extract.LC 50 s values of L 2 larvae calculated from equations of regression lines (illustrated on Figure 4) were 3.57, 2.08 and 1.5 mg/ml for infused, macerated aqueous extract and ethanolic extract respectively.Like on eggs, distilled water and 1.5% DMSO had no effect on larvae while M. oleifera extracts were active in a concentration dependent manner.

Discussion and Conclusion
In developing countries, the identification of a plant with anthelminthic property may help to build an integrated and sustainable approach for the control of gastro-intestinal nematodes in small ruminants.The objective of this study was to evaluate in vitro ovicidal and larvicidal efficacy of aqueous and ethanolic leaf extracts of M. oleifera against H. contortus.In vitro bioassay provides means to rapidly screen for plant extracts and to analyze the possible mechanisms involved in the interactions between active compounds and parasites [19].
Asase et al. [20] reported that in vitro tests using free living stages of parasitic nematodes as is the case in the present study are considered the best means of screening the anthelminthic activity of new plant compounds.In this study, M. oleifera leaf extracts presented a concentration-dependent activity against the four different free-living stages of H. contortus, suggesting that increase in concentration of plant extract is followed by a supplementary input of different active compounds.Infused aqueous extract and ethanolic extract of M. oleifera presented comparable activity on eggs.The first extract (infused) inhibited 69% ± 1% and 97.9% ± 4.2% of egg embryonation and egg hatch at 5 mg/ml with IC 50 s values of 1.52 and 1.75 mg/ml respectively.While the latter (ethanolic) inhibited 92.8% ± 6.2% and 99% ± 2% of egg embryonation and egg hatch at 5 mg/ml, with IC 50 s values of 0.985 and 1.74 mg/ml respectively.Tatik and Dwatmadji [21] recorded the same observation between crude aqueous and ethanolic extracts of Melastoma malabatricum on eggs of H. contortus.They also found out that there was no statistically significant difference in the activity of aqueous and hydro-alcoholic extracts of Hedera helix after evaluation of in vitro anthelminthic activity against H. contortus.The observed uniform activity of infused aqueous extract and ethanolic extract of M. oleifera on eggs could be due to the presence of similar or related chemicals having ovicidal property in nearly equivalent proportions.Based on LC 50 s values, ethanolic extracts presented the highest larvicidal activity since it registered the lowest values of 1.89 and 1.5 mg/ml on L 1 and L 2 larvae respectively.These LC 50 s values revealed that L 1 larvae were more susceptible to ethanolic extract than L 2 larvae, confirming the literature findings of Soulby [22] and could be explained by the fact that since L 2 are just from the process of molting, they are still weak and more vulnerable to active compounds [23].However, Wabo Poné et al. [24] found opposite results when evaluating in vitro activities of acetonic extracts of leaves of three forage legumes on H. contortus, since they obtained LC 50 s values below 0.9 and above 1 mg/ml for L 1 and L 2 larvae respectively.Infused aqueous extract generally exhibited higher activity on eggs than on larvae.Macerated aqueous extract showed a good activity on almost all the four stages of parasites.The probable reason for the minor differences between aqueous and ethanolic extracts could be due to variation in solubility of the active compounds in the solvent.The ovicidal and larvidal activities observed in this study with different extracts may be attributed to the presence of saponins, steroids, carbohydrates, alkaloids, tannins, flavonoids which were previously reported to be present in leaves of M. oleifera after preliminary phytochemical screening [25].These secondary metabolites may take the two ways of anthelminthic drugs that are diffused through egg shells or cuticle of larvae or diffusion into intestinal cells to exert their inhibition or mortality action on eggs and larvae of H. contortus.
In conclusion, the activity from this study shows the potential value of Moringa oleifera leaf extracts in the management of haemonchosis, since inhibition of egg embryonation, egg hatch and mortality of L 1 and L 2 larvae are important in reducing pasture contamination thereby helping in the overall helminth control programme.However, in order to use this plant for better beneficial purpose in the fight against gastro-intestinal nematodes, further in vivo and toxicity studies are necessary.

Table 2 .
Mean inhibition percentage of egg hatch ± standard deviation of Moringa oleifera leaf extracts at different concentrations against Haemonchus contortus.

Table 4 .
Mean mortality percentage of L 2 larvae ± standard deviation of Moringa oleifera leaf extracts at different concentrations against Haemonchus contortus.