Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs

Background: Fasciolosis is an important zoonotic disease affecting the productive performance of farm animals in Egypt. Aim: The aim of the present study was comparing the ovicidal effect of different extracts as an alcoholic (Methanolic and Ethanolic) and aqueous Moringa oleifera leaf extracts on Fasciola gigantica non-embryonated and developed eggs. Materials and Methods: Tested concentrations of extracts ranged from 12.5 to 800 mg/ml. Nitroxynil was used as reference drug with a dose of 100 mg/ml. Results: M. oleifera alcoholic and aqueous extracts showed a concentration-dependent ovicidal effect on F. gigantica non-embryonated and developed eggs. Based on LC50 values, water extract showed the highest ovicidal activity since it registered the lowest values of 2.6 mg/ml on non-embryonated eggs. Non-embryonated eggs were more susceptible to aqueous extract than developed eggs. On the other hand, the developed eggs were more susceptible to ethanolic extract than non-embryonated eggs even the lowest LC50 (12.38 mg/ml). Conclusion: M. oleifera leaf extracts especially aqueous extract could be a promising step in the field of controlling fascioliasis. Further, in vivo studies are needed to enlighten the therapeutic potential of M. oleifera extracts in treating F. gigantica infection.


Introduction
Fasciolosis is considered one of the most important helminthic diseases affecting livestock in many countries around the world. It is commonly found in sheep and cattle [1]. Bovine fasciolosis usually had no visible clinical signs. However, if the cattle infection was chronic, it might cause weight gain loss, reduction in the milk yield, and fertility problems [2,3].
Many available chemotherapeutic anthelmintics had side effects on the host, and it is necessary to decrease the use of these drugs for parasitic control, not only for their resistance but also because of growing concerns about the adverse consequences on the ecosystem. Naturally produced plant anthelmintics offer an alternative that overcomes some of these effects and is both sustainable and environmentally acceptable [4]. Around the world, new plants having medicinal properties against parasites of ruminants had been explored, and they had shown good results [5]. Moringa oleifera is considered as one of the most useful trees for the reason that every part has many beneficial properties [6]. M. oleifera is commonly known as Drumstick and contains many active compounds such as alkaloids, flavonoids, saponins, tannins, and triterpenoids [7]. Extracts from this plant have several pharmacological effects such as anthelminthic [8][9][10], anti-inflammatory, antimicrobial, and antioxidant [11].
The aim of the current study was to compare the ovicidal effect of different extracts as an alcoholic (methanol and ethanol) and aqueous M. oleifera leaf extracts on Fasciola gigantica non-embryonated and developed eggs.

Ethical approval
F. gigantica eggs were recovered from gallbladders of cattle and buffaloes slaughtered in the government abattoir according to governmental regulations.

Preparation of extracts
Alcoholic extract 150 g of M. oleifera green leaves were separately extracted in 1500 ml of 80% ethanol (ethanolic extract) and 80% methanol (methanolic extract), then manually shaken for 30 min and allowed to stand with continuous shaking at a shaking water bath for 3 days. After that, solutions were filtered with sterile filter papers (Whatman No.1) into a clean conical flask and dried under reduced pressure in a rotary evaporator at a temperature below 50°C and stored at 4°C until use.

Aqueous extract
10 g of M. oleifera shade dried leaves were ground into powder and extracted by maceration in 1000 ml of double distilled water accompanied by stirring for 24 h in cold conditions 4°C then filtered with sterile filter papers (Whatman No.1) into a clean conical flask and stored at −20°C until use.

Collection of F. gigantica eggs
F. gigantica eggs were recovered from bile of the gallbladders of infected cattle and buffaloes slaughtered in Cairo local abattoir, Egypt. Gallbladders were washed and examined individually. Each gallbladder was evacuated separately in the 1 L cylinder, mixed with tap water, left to sediment and then the supernatant was decanted without disturbance of the sediment. This process was repeated 3-5 times. The clean F. gigantica eggs were collected after sedimentation and stored in distilled water to be used as fresh as possible.

Ovicidal effect of M. oleifera on F. gigantica eggs
Two experiments were involved in the present study; one was for non-embryonated and the other was for developed (morula stage) F. gigantica eggs. The alcoholic extracts concentrations were 12.5, 25, 50, 100, and 200 mg/ml, while in aqueous extract they were 100, 200, 400, and 800 mg/ml. Three replicates of every dilution were prepared in each treatment. Each replicate was contained 10 ml double distilled water and about 100 F. gigantica eggs and the selected extract concentration. Three replicates of 100 mg/ml nitroxynil drug were tested. Another group of eggs were incubated in distilled water and served as control group. The Petri dishes were incubated at 28°C in dark environment for 14 days. The numbers of hatched and non-developed eggs were counted. F. gigantica eggs and their developmental stages including the morula, eyespot stage or embryonated eggs and hatched eggs were identified as previously described [12].
The percentage of hatched and developed eggs for each treatment was calculated [13], and hatching ratio was calculated according to Canevari et al. [14].

Statistical analysis
Data of ovicidal effect of different treatment groups were analyzed for the means and standard deviations. The significance of the results was evaluated using independent sample t-test and analysis of variance and Duncan test using Statistical Package for the Social Sciences computer programs [15].

Results
The ovicidal effects of the different M. oleifera extract on the F. gigantica eggs are displayed in Tables-1 and 2. M. oleifera alcoholic and aqueous extracts, as well as nitroxynil, showed an ovicidal effect on Fasciola non-embryonated and developed eggs (Figures 1-6). The effect of these extracts on eggs was concentration dependent. There were no statistically significant differences between the ovicidal activity of M. oleifera methanolic and ethanolic extracts in non-embryonated eggs. Water extract at a concentration of 800 mg/ml had the same effect on eggs as 100 mg/ml methanolic and ethanolic extracts. In the developed eggs experiment, the highest concentrations of the three extracts were statistically the same.
Regarding the comparing the ovicidal activity of the three extracts on the eggs with independent sample t-test, significant differences were found between the 2 stages of eggs development at almost all concentrations of methanolic and aqueous extracts. Almost all concentrations of ethanolic extract had the same effect on non-embryonated and developed eggs (Table-3).
LC 50 of the different concentrations was calculated as shown in Table-4. LC 50 was higher in non-embryonated eggs than developed eggs for methanolic and ethanolic extracts. Water extract exhibited a different effect as LC 50 of aqueous extract on developed was higher than that of non-embryonated eggs.

Discussion
In the current study, M. oleifera leaf extracts presented concentration-dependent activities against the two developmental stages of F. gigantica eggs tested. These results are similar to those obtained by Pessoa et al. [16] against Haemonchus contortus by the essential oil of Ocimum gratissimum. Furthermore, they confirmed the results obtained previously on rhabditiform larvae of Ancylostoma caninum by Poné et al. [17] and on eggs of the nematode A. caninum with the extracts of the shrub Canthium mannii by Poné et al. [18].
Alcoholic and aqueous extracts of M. oleifera represented high anthelmintic activity on eggs. In addition, all extracts rendered most of non-embryonated F. gigantica eggs undeveloped suggesting that the bioactive compounds were lethal to the blastomeres. Furthermore, Rastogi et al. [8] found that the ethanolic extract of M. oleifera showed strong anthelmintic activity at 25, 50, and 100 mg/ml against Indian earthworm. On the contrary, M. oleifera did not give satisfactory in vitro anthelmintic results on Ascaris suum with Peter and Deogracious [19] with a median effective dose more than 50 mg/ml in the initial screening.
The ovicidal activities observed in the present study using different extracts might be attributed to the presence of saponins, steroids, carbohydrates, alkaloids, tannins, and flavonoids which were     reported in M. oleifera leaves [20,21]. Previously, it was suggested that tannins and saponins possibly penetrated the egg affecting the morula [22] and stop hatching [23] of H. contortus eggs.
In the current study, there were no statistically significant differences between ovicidal activity of M. oleifera methanolic and ethanolic extracts in non-embryonated and developed eggs. The probable reason for the minor differences between aqueous and alcoholic extracts could be due to variation in solubility of the active compounds in the solvent. Infused aqueous extract and ethanolic extract of M. oleifera tested by Tayo et al. [24] on H. contortus, in vitro, presented comparable activity on eggs. The observed similar activity of ethanolic extract and infused aqueous extract of M. oleifera leaves on eggs could be due to the presence of similar or related chemicals having ovicidal properties in approximately equivalent proportions. On the contrary, Poné et al. [18] found that the ethanol extract was more potent on the eggs when compared to hot and cold water extracts.
Based on LC 50 values, water extract presented the highest ovicidal activity since it registered the lowest values of 2.6 mg/ml on immature non-embryonated eggs suggesting that non-embryonated eggs were more susceptible to aqueous extract than developed eggs. On the other hand, developing eggs were more susceptible to ethanolic extract than non-embryonated eggs as ethanolic extract had the lowest LC 50 (12.38 mg/ml). These results agreed with Tayo et al. [24] who found All data expressed as mean±SD. Means followed by different letters indicated significance. SD=Standard deviation  [18] found that LC 50 value of the ethanol extract of C. mannii was relatively low, indicating that this extract is more active on non-embryonated eggs than aqueous extracts. This conflict might be due to they used H. contortus eggs, and in the current study, F. gigantica eggs were used. Although not quite exact on how M. oleifera inhibit egg embryonation, Sreelatha et al. [25] explained that M. oleifera leaf extract could induce cellular apoptosis, morphological change and DNA fragmentation in a type of human cancer cell.

Conclusion
The current study introduced M. oleifera leaf aqueous extract which offered a promising ovicidal effect on F. gigantica eggs. The future scope involves the need of in vivo study to enlighten the therapeutic potential of M. oleifera extracts in treating F. gigantica infection. Furthermore, isolation of phytoconstituents responsible for these activities is required.

Authors' Contributions
This study was designed and supervised by AGH. While, SEH, EEE and DA carried out extracts preparation. AMM and NIT collected the samples. KNA, SEH, EEE and DA examined gallbladders. KNA, SEH, DA and EEE carried out the application of plant extracts on eggs. DA analyzed and interpreted the data. All authors have prepared, read and approved the final manuscript.

Acknowledgments
This work was financially supported by the project number 11020201 entitled: "Evaluation of