European Journal of Experimental Biology Open Access

Keywords

Haemonchus contortus; Resistance; Ziziphus mucronata; Phytochemical; Inhibition

Introduction

Helminths play a significant role in causing helminthosis in small ruminants leading to enormous economic losses. These helminths occur worldwide and cause losses which are associated with among others reduced milk production, lowered weights and mortality [1].

Haemonchosis is a condition caused by Haemonchus contortus which has been listed among the most important conditions affecting production of small ruminants especially sheep and goats in developing countries [2].

Control of these parasites in ruminants basically depends on routine use of anthelmintic drugs. However, in recent years, the efficacy of these anthelmintic have decreased due to development of resistance by parasites [3]. This has awakened the interest of herbal medicinal plants as an alternative source for control of these parasites [4,5].

Ziziphus mucronata plant, also known as buffalo thorn, is a medium-sized with spreading canopy. In Africa, extracts from the plants are used for treatment of variety of conditions including gastro-infections, schistosomiasis, boils, chronic cough, infertility, oedema, pneumonia, snake bite, toothache, venereal diseases and wounds [6-8].

A study [9] showed that extracts from Z. mucronata have antischistosomiasis properties. In Zimbabwe, the leaves and fruits are sources of feed for small ruminants [10].

The present study was carried out to evaluate in vitro ovicidal and larvicidal activity of aqueous and methanolic extracts of Z. mucronata barks against H. contortus egg and larval stages isolated from sheep.

Materials and Methods

Collection and preparation of plants

Ziziphus mucronata (local name in Chad: Ngohkroh) barks (500 grams) were collected from Deli location in the southern region of Chad during the month of April 2015. The plant materials were identified and authenticated by botanists from Herbal Treatment Centre in Deli, Chad.

The collected plant materials were shade dried for three weeks at ambient temperature, ground to fine powder by using pestle and mortar. The powdered bark was packaged into envelope and transferred to Kenya. Extraction has been done at Biochemistry and Chemistry Laboratories of Jomo Kenyatta University of Agriculture and Technology.

Aqueous extraction

Aqueous extraction of Z. mucronata barks were prepared according the procedures described by [11]. Briefly, 200 grams of fine powder were weighed and mixed with 1000 ml of distilled water in 2 Litres flask and boiled for 2 hours. It was then cooled to 40°C and the residues filtered using Whatman filter paper No 1. The extracts were dried in a freeze dryer (Christ Alpha 1-4 LD, SciQuip Ltd, Newtown UK) and stored at 4°C until required for in vitro anthelmintic activity described below.

Methanolic extraction

The fine ground powder of Z. mucronata barks were weighed, extracted with methanol and residues filtered using Whatman filter paper No.1. The extracts were concentrated using a rotary evaporator (BUCHI R-200, Labortechnik AG CH-9230 Flawil Switzerland) at 45°C and stored at 4°C until required for anthelmintic activity.

Phytochemical screening

Aqueous and methanolic extracts of Z. mucronata barks were subjected to phytochemical screening using standard procedure described previously [12].

In vitro anthelmintic activity

In vitro anthelmintic activity was conducted according to World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines [13] with slight modification on parasite collection, eggs preparation, egg hatch and larval mortality assays as described below:

Parasite collection and eggs preparation

Mature adult parasites of H. contortus were collected directly from abomasum of slaughtered sheep from a local slaughter house in Ruiru, Kenya. The worms were transported to the laboratory in Phosphate Buffer Saline (PBS, pH 7.4).

Identification and separation of adult mature female were done using a microscope using the method [14]. They were washed using PBS and crushed using pestle and mortar in 5 ml PBS to liberate eggs. McMaster slide technique, as described [15], was used to estimate the concentrations of egg in aliquots of 200 μl.

Egg hatch inhibition (EHI) assay

For each extract, a stock solution of 4 mg/ml was taken as the initial concentrations and serial dilutions of 0.0625 to 4 mg/ml prepared in phosphate buffered saline. Approximately 100 H. contortus eggs in 200 μl of egg suspension were pipetted into each well of 96 well microtitre plates.

A total of 200 μl of Z. mucronata extracts were then added into each well. Albendazole (Albendazole, Sigma-Aldrich) was used as positive control while untreated eggs in PBS were used as negative control. Three replicates for each concentration of both aqueous and methanolic extracts of Z. mucronata and controls were performed. The plates were labelled and incubated for 48 hours at 27°C and 70% relative humidity. After 48 hours of incubation, hatched larvae (live or dead) and unhatched eggs were counted using a compound microscope at 40X magnification with the help of a counter [16,17]. The egghatch inhibition rates assessed were calculated by the following formula:

Larval mortality assay (LMA)

Larval mortality assay using larvae were performed according to [18]. After collection of adult parasite, the female H. contortus was identified and separated from male as described above. Eggs were recovered by grinding the female with pestle and mortar in 5 ml PBS. One hundred eggs in 180 μl of egg suspension were put into each well of 96 well microtitre plates. A 20 μl of nutritive media (comprising of 1 g yeast in 90 ml of normal saline and10 ml Earle’s balanced salt) was added into each well. The plates were then incubated under humidified condition at ambient temperature for 48 hours. After 48 hours of incubation, 200 μl of Z. mucronata extracts in concentrations ranging from 0.0625 to 4.0 mg/ml were added to respective plates. Albendazole (Albendazole, Sigma-Aldrich) was used as positive control while untreated larvae in PBS were used as negative control.

There were three replicates for each extract concentration and control. The plates were further incubated for 24 hours (total of 3 days). Counting of all larvae in each well was done under an inverted microscope. The percentage of mortality of the larvae was determined using the following formula:

Statistical analysis

Mean percentage egg hatch inhibition rates and larval mortality from Z. mucronata extracts and Albendazole at different concentrations were compared using one-way ANOVA test at p<0.05 significant levels. The inhibition concentration required to inhibit 50% (IC₅₀) for ovicidal and effective concentration (EC₅₀) values for larvicidal activity were determined using the regression line of probit according to the log10 of the extract concentration.

Results

Extraction and phytochemical screening

The per cent yield for methanol extraction was higher (4.5 ± 0.21%) than that of aqueous extraction (2.6 ± 0.5%).

The results for phytochemicals screening of Z. mucronata extracts are presented in Table 1. The phytochemicals present found in the extracts included saponins, tannins, glycosides, flavonoids and steroids.

Phytochemical	Aqueous extracts	Methanol extracts
Saponins	+	+
Tannins	+	+
Alkaloids	-	-
Glycosides	+	+
Flavonoids	+	+
Steroids	+	+

Table 1 Phytochemical screening of aqueous and methanolic extracts Ziziphus mucronata. Key:‘-’ Absent, ‘+’ Present.

Egg-hatch inhibition (EHI) assay

Different concentrations of aqueous and methanolic extracts of Z. mucronata barks in concentrations ranging from 0.0625-4 mg/ml were tested for their anthelmintic activity and results presented in the Figure 1.

Figure 1: Mean hatch inhibition for eggs of Haemonchus contortus exposed to Ziziphus mucronata extracts of and Albendazole.

The higher drug concentration resulted in higher egg hatch inhibition compared with lower concentration suggesting a concentration dependent response.

At all extracts concentrations, the egg hatch inhibition was highest in eggs exposed to Albendazole, followed by methanol and aqueous extracts. There was no significant (p>0.05) difference in egg hatch inhibition by aqueous and methanolic extracts of Z. mucronata. However, there were significant (P<0.05) differences in egg hatch inhibition of extracts compared to that Albendazole.

The concentration required to inhibit 50% (IC₅₀) of both extracts and Albendazole are presented in Table 2. Methanolic extract showed a higher activity with IC₅₀ value of 3.9 mg/ml as compared with aqueous extract with IC₅₀ value of 14.7 mg/ml.

Sample type	Mean IC50 values (mg/ml)	Lower bound	Upper bound
Aqueous extract	14.683	6.896	55.000
Methanol extract	3.932	2.416	8.282
Albendazole (Positive control)	0.050	0.024	0.079
95% confidence limits for concentration (mg/ml)

Table 2 IC₅₀ values of methanol and aqueous extracts of Z. mucronata barks and Albendazole exposed to eggs of H. contortus

Larval mortality assay (LMA)

Results of larval mortality and concentration required to inhibit 50% of infective larvae are presented in Figure 2 and Table 3, respectively.

Sample type	IC50 values (mg/ml)	Lower Bound	Upper Bound
Water barksextract	7.516	3.638	28.032
Methanol Barks extract	2.646	1.593	5.929
Albendazole (Positive control)	0.045	0.031	0.057
95% confidence limits for concentration (mg/ml)

Table 3 IC₅₀ of methanolic and aqueous extract of Z. mucronata exposed to larval stages of H. contortus

Figure 2: In vitro larvicidal activity of methanolic and aqueous extracts of Ziziphus mucronata barks.

Aqueous and methanolic extracts of Z. mucronata in different concentration and ratios showed variable effect on mortality of larvae of H. contortus.

At the maximum tested concentration of 4 mg/ml, the highest (53.3 %) larval mortality was from methanolic extracts with while the least (43.3%) was from aqueous extract.

Albendazole required a maximum concentration of 0.5 mg/ml to induce 100% larval mortality. There was a significant difference in activity among different drug/extracts concentration (p<0.05).

The highest EC₅₀ value against larval mortality was revealed by Albendazole (0.045 mg/ml), followed by methanolic extract (2.7 mg/ml) and least was aqueous extract (7.5 mg/ml). The EC₅₀ were significantly (p<0.05) different from each other. Further, the IC₅₀/EC₅₀ for larval mortality was significantly (p<0.05) higher than that of egg hatch inhibition.

Discussion

Haemonchus contortus causes significant losses in small ruminant production. Farmers in low resource settings in Africa rely on herbs to treat helminths but their efficacy has not been well evaluated. Further the phytochemicals found in these plants have also not been investigated. In the current study, the phytochemicals and efficacy of Z. mucronata were investigated. The phytochemical screening in the present study showed the presence of various phytochemical constituents such as flavonoids, saponins, steroids, tannins and glycosides. These significant variations in the phytochemical contents of a plant part are due to number of environmental factors as previously mentioned [19]. These phytochemicals account for their medicinal value. For instance, tannins extracted from plants have been shown to have anthelmintic, antidiarrhoeal and antimicrobial activities [20,21], while Saponins have anthelmintic, antidiarrhoeal and anticancer properties [22-24]. Glycosides and Steroids produced by the plant extracts are known for their antidiarrhoeal action [24,25], whereas Flavonoids in plants have been shown to have antidiarrhoeal and antimicrobial activities [22,26].

The current study evaluated the efficacy of bark extracts of Z. mucronata against egg and larval stages of H. contortus. The egg hatch assay was initially developed for the diagnosis of benzimidazole resistant helminths. This test has, however, been used for screening of plants compounds for their anthelmintic activity [11,27,28]. In this study, Methanolic extract showed a higher activity with IC₅₀ value of 3.9 mg/ml as compared with aqueous extract with IC₅₀ value of 14.7 mg/ml. This could be attributed to the difference in proportions of active components that were responsible for anthelmintic activity. A previous study [29], on aqueous extracts of Hedera helix showed IC₅₀ value of 0.12 mg/ml respectively when tested against H. contortus egg. Aqueous extract of Coriandrum sativum had an IC₅₀ of 0.12 mg/ml while the hydroalcoholic extract had an IC₅₀ of 0.18 mg/ml on egg hatch inhibition test [30]. Thus, these plants had a relatively higher activity compared with Z. mucronata aqueous extract. Albendazole showed comparable results with that of in vitro anthelmintic activities of four Ethiopian medicinal plants against H. contortus which had IC₅₀ value of 0.04 mg/ml [30], a value which is close to that reported in the current study. In the present study, aqueous and methanolic extracts of Z. mucronata in different concentration and ratios showed variable effect on mortality of larvae of H. contortus.

Similar to egg inhibition, the highest EC₅₀ value against larval mortality was by Albendazole (EC₅₀=2.7 mg/ml) and least was aqueous extract (EC=7.5 mg/ml). Similar results by a previous study [31] showed that the EC₅₀ aqueous extract of Fumaria parviflora against H. contortus was 10.23 mg/ml. Comparable results on larval mortality showed EC₅₀ values of 15.14 and 12.88 mg/ml for aqueous and ethanolic extract of Adhatoda vasica, respectively [32]. Findings of this study showed that increasing the concentration of the plant extracts resulted in increased anthelmintic activity. A similar observation have also been made by a previous study [33] which demonstrated that the receptors get saturated with increasing concentration of active ingredient. It is likely that at higher concentration all binding receptors on the larvae were occupied thus leading to hyper-polarisation of membranes limiting excitation and impulse transmission causing flaccid paralysis of larvae muscles, a similar observation also made by previous study [34]. The present study showed that larval development inhibition by the extracts showed a better activity in contrast with egg hatch inhibition. The possible explanation could be due to the difference in structure of the egg shell and cuticle of larvae of H. contortus through which absorption of chemicals take place [30].

Conclusion

The findings of this study showed that, aqueous and methanolic extracts of Z. mucronata have a potential anthelmintic activity on eggs and larvae of H. contortus parasite. It is postulated that saponins and tannins identified could be responsible for the observed anthelmintic activity. Therefore, extracts from this plant can be further assessed in order to develop novel anthelmintic drug for control of H. contortus and other nematodes affecting livestock and man. Further studies geared towards isolation, identification, characterization and purification of bioactive compounds from this plant should be carried out in order to increase its efficacy.

Acknowledgements

The authors are thankful to African Union and Japan International Cooperation Agency (JICA) for funding the project. Special thanks also to Pan African University Institute for Basic Sciences, Technology and Innovation (PAUISTI) and Jomo Kenyatta University of Agriculture and Technology (JKUAT) for providing technical support.

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