In vitro evaluation of anthelmintic activity of tannin-containing plant Artemisia extracts against Haemonchus contortus from goat

Karim, M.A., Islam, M.R., Lovelu, M.A., Nahar, S.F., Dutta, P.K. and Talukder, M.H. 2019. In vitro evaluation of anthelmintic activity of tannin-containing plant Artemisia extracts against Haemonchus contortus from goat. Journal of Bangladesh Agricultural University, 17(3): 363–368. https://doi.org/10.3329/jbau.v17i3.43216 J Bangladesh Agril Univ 17(3): 363–368, 2019 https://doi.org/10.3329/jbau.v17i3.43216


Introduction
Gastrointestinal parasitism has been identified as one of the major constraints that hinder the development of livestock population (Kakar and Kakarsulemankhel, 2008) and adversely affects the health and productivity of animals.Despite significant losses by gastrointestinal parasitism, the problems are often neglected and overlooked as majority of the infected animals show a number of obvious clinical signs throughout their productive life and their effects are gradual and chronic (Raza et al., 2010).Among the parasitic diseases, gastrointestinal nematodes such as Haemonchus contortus, Trichostrongylus spp., Cooperia spp., Oesophagostomum columbianum, Trichuris spp.and Strongyloides papillosus are most common in Bangladesh (Rahman and Mondal, 1983).The overall prevalence of gastrointestinal helminths in goat is 63.41% in Bangladesh (Hassan et al., 2011).Overall 65.63% goats are infected with H. contortus in Bangladesh (Shahiduzzaman et al., 2003).Grazing animals are most susceptible to gastrointestinal nematodes (GINs) infection than stall fed animals.GINs cause significant production loss in grazing ruminants.Adult stomach worm, H. contortus is a blood-sucking abomasal helminth of small ruminants responsible for major economic losses to producers worldwide.Furthermore, frequent uses of anthelmintics are increasing resistance at an alarming rate (Dyary, 2016).
In several developing countries, small farmers have limited access to commercial anthelmintics and veterinary services due to their non-availability and/or to their high cost.These stockowners rely on the ethno-veterinary medicine as an alternative and a sustainable control option readily adaptable (Hussain et al., 2008;Sindhu et al., 2010).For these various reasons, interest in the screening of medicinal plants for their anthelmintic activity remains of great scientific interest despite the extensive use of synthetic chemicals in modern clinical practices all over the world (Akhtar et al., 2000).The Indian subcontinent mainly Bangladesh, Pakistan and India bound with plants that were known to have medicinal properties and have been used for their curative powers throughout the ages.Recently, much interest in the field of medicinal plants has started throughout the world and many countries have already come to realize not only potential as means of alleviating health problems but also their economic value (de Padua and Pancho, 1983).
The condensed tannin (CT) is a diverse group of polymeric flavonoids of tanniniferous plant origin that readily form complex with carbohydrates and improve metabolism in ruminants.Dietary supplementation of CT through forages, tropical tanniniferous tree leaves, skin and fruit have been found effective against different developmental stages of GIN parasites.H. contortus species was used by several scientists to evaluate the anthelmintic effects of various medicinal plant species (Alawa et al., 2003;Assis et al., 2003;Eguale et al., 2007).In vitro assays are relevant and cheaper than in vivo methods.Egg hatch assay (EHA) is currently used for the detection of anthelmintic resistance in GIN (Timothy et al., 2012).Suitable environment is required to perform in vitro assays of egg hatching, larval and adult worm motility of GINs.Higher concentration of GIN larvae occur at the temperature between 20 and 30ºC and rainfall between 31.9 and 122.7 mm.(Shahiduzzaman et al., 1999).
There are a number of research works on the anthelmintic effects of medicinal plants in Bangladesh (Mostofa et al., 1983;Begum et al., 1997, Shahiduzzaman et al., 2005).In this context, investigations on tannin-containing plants such as Artemisia species (Ashok and Kumud, 2010), Terminalia arjuna, Fumaria perviflora, Moringa oleifera, Butea monosperma, etc (Bauri et al., 2015) might contribute to develop effective but low-cost herbal anthelmintic.However, these are mostly used in crude forms and their pharmacological preparations, dosages and mode of actions are not based on strong scientific evidence.Tannin-containing plant A. vulgaris is very much available in our country in and around the crop field, road side, bush, forest etc.There is a great opportunity to utilize this plant to minimize stomach worm of ruminants in Bangladesh.To our best knowledge, there is no published article on the anthelmintic activities of tannin-containing plants against stomach worm in Bangladesh.Therefore, this research work may provide useful information on anthelmintic activities of tannin containing plant Artemisia to use against stomach worm of ruminants

Collection and processing of plant Artemisia
The aerial part of the tannin-containing plant Artemisia vulgaris was collected from BAU campus and its surrounding rural areas and brought to the laboratory of the Department of Parasitology.After collection, the plant materials were rinsed under running tap water and cut into small pieces.Firstly the plant materials were dried in shade and then they were dried in the oven at 55-60°C until gain a constant weight.

Preparation of dust, crude extract and stock solution
Dust was prepared by pulverizing the dried plant materials and preserved these into an airtight plastic container till their use in extract preparation.Prepared plant dusts were used for preparation of plant extract and finally condensed extracts those were preserved in tightly corked-labeled bottle and stored in refrigerator until use (Akkari et al., 2014).Stock solutions of plant extracts were prepared by diluting the condensed extracts with (PBS).Different concentrations i.e. 100 mg/ml, 50 mg/ml, 25 mg/ml of each category of plant extracts were prepared by dissolving them in the PBS prior to anthelmintic screening to use at concentrations 20 mg/ml, 10 mg/ml, 5 mg/ml, respectively.

Collection of adult worms, eggs and cultivation of larvae (L3) and their maintenance
The abomasum, small and large intestines of goats slaughtered in the local markets were collected and brought to the laboratory.They were opened in a plastic bucket separately and the contents were washed in tap water.The process was repeated for several times until the sediment becoming transparent.Then the adult stomach worms were collected in a petridish containing PBS and then kept in incubator at 38°C until required for experiment on the same day.The collected worms were washed for several times with PBS.Then uteri of gravid females were dissected out, crushed gently in a petridish.As a result eggs were released in the PBS solution.An in vitro culture system has already been established in our laboratory for harvesting the L3 stage larvae of GINs (Shahiduzzaman et al., 1999).A required volume of PBS was added to eggs and incubated at room temperature (25-30°C) for about 72 hrs transferred to a 100 ml beaker and incubated further until development of L3.During cultivation the culture media were monitored every morning for observing the development.After development of L3, the culture media were washed several times in PBS through centrifugation at 2000 rpm for 7 minutes and finally counted and suspended in a 100 ml beaker.After cultivation, the infective larvae were maintained in the laboratory by incubating them at 25-30°C and in sterile condition.

In vitro test of crude aqueous and ethanolic extracts for anthelmintic activity
Examination of eggs for development of larvae Different concentration of crude aqueous and ethanolic extracts of Artemisia were used for treatment.Three concentrations (100 mg/ml, 50 mg/ml, and 25 mg/ml) for each extracts (stock solution) were used with three replicates of each.Untreated eggs in PBS with 0.5% Dimethyl sulf-oxide (DMSO) solution were used as negative control with three replicates.For aqueous extracts, every 1 ml stock solution was placed in each test tube containing approximately, 200 eggs in 4 ml of PBS.After placing stock solution, each tube was then contained 5 ml with the concentration of 20mg/ml, 10mg/ml, and 5mg/ml of aqueous extracts respectively.For ethanolic extracts, at the same concentrations, 1 ml stock solution in 4 ml PBS with 0.5% DMSO was used.The test tubes were covered and incubated at room temperature (25-30°C) for 72 hours.Hatched larvae (dead or alive) and unhatched eggs were counted under dissecting microscope at 40x magnification and calculated (Akkari et al., 2014).

Detection of larval motility
Untreated Larvae in PBS with 0.5% DMSO solution were used as negative control with three replicates.For aqueous extracts, every 1 ml stock solution was placed in each petridish containing 100 larvae (L3) in 4 ml of PBS.Each petridish was then contained 5 ml with the concentration of 20mg/ml, 10mg/ml, and 5mg/ml of aqueous extracts respectively.For ethanolic extracts, at the same concentrations, 1 ml stock solution in 4 ml PBS with DMSO (0.5%) was used.All the tests were performed in triplicates.The petridishes were covered and incubated at room temperature (25-30°C).Inhibition of larval motility was the criteria of anthelmintic activity.The required delays for larvae paralysis and/or complete immobility were recorded at 0; 2; 4; 8; 16 and 24 hours.To test if the larvae could retrieve their motility after 24 h, they were washed with distilled water and resuspended in PBS for 30 min.Worms death was ascertained by the absence of motility during an observation period of 5-6 sec.Motile (live) or non-motile (dead) larvae (L3) were counted under dissecting microscope at 40x magnification and calculated (Akkari et al., 2014).

Detection of adult worm's motility
Untreated Adults in PBS with 0.5% DMSO solution were used as negative control with three replicates.Similarly, for aqueous extracts, every 1 ml stock solution was placed in each petridish containing 5 adult worms in 4 ml of PBS with same concentration.An exactly similar protocol was applied to test L3 stage and adult worm's motility for ethanolic extracts (Akkari et al., 2014).

Statistical analysis
In vitro effects of different concentration of Artemisia extract were statistically analyzed with ANOVA technique to obtain the level of significance using MSTAT-C package program developed by Russel (1986).The mean differences were compared by Duncan's New Multiple Range Test (DMRT) (Gomez and Gomez, 1984).

Egg hatch assay
In this study, ethanolic extracts showed higher inhibitory effects than aqueous extract on egg hatching.In ethanolic extracts, none of the eggs were hatched at each of the 5 mg/ml, 10 mg/ml and 20 mg/ml concentration whereas in aqueous extracts, none of the eggs were hatched at the concentration of 20 mg/ml and 10 mg/ml but 25% eggs were hatched at the concentration of 5 mg/ml.The 5 mg/ml crude ethanolic extracts completely (100%) inhibited egg hatching whereas10 mg/ml of crude aqueous extracts (Table 1) did.These findings could also be compared with Akkari et al. (2014) who recorded the findings that the maximum concentration required to induce complete (100%) egg hatch inhibition for crude aqueous and ethanolic extracts was 2 mg/ml.Thymus capitatus (another important pastoral species form arid Tunisia) aqueous and ethanolic extracts totally inhibited H. contortus egg hatching at 2 mg/ml concentration (Landolsi et al., 2013).

Adult worm's mortality assay
In case of adult worm's mortality assay, after 2, 4, and 8 hours of exposure, the crude ethanolic extract induced 53.33%, 73.33% and 100% mortality at tested 20 mg/ml concentration respectively, (Table 4) while the crude aqueous extract induced 47.67%, 60% and 86.67% at the same concentration, respectively (Table 5).These findings could also be compared with Akkari et al. (2014) who recorded the findings of Artemisia campestris crude extracts against H. contortus that induced 91.3% and 100% mortality at the tested concentration of 2 mg/ml after 8 and 24 h of exposure of the crude ethanolic extract, respectively, whereas the crude aqueous extract induced 3.22% and 70.96% at the same concentration, respectively.
This study revealed that all three different concentrations of crude ethanolic and aqueous extracts have in vitro anthelmintic effects against eggs, larvae and adult H. contortus.However, in vitro anthelmintic effects of several plants have been demonstrated against stomach worm of goats in various parts of the world (Akhtar et al., 2000;Dano and Bogh, 1999).In vitro screening of both crude aqueous and ethanolic extract are more or less similar but ethanolic extract showed more anthelmintic activity against both larvae and adult worms (Sisay et al., 2012).
In this study, it was found that in all cases, the complete inhibition of egg hatching, larval motility and adult worm mortality depends on the type and concentration of the extracts used (Tables 1, 2, 3, 4 & 5).The time for complete inhibition in motility of larvae and mortality of adult decreased as the concentrations of these crude extracts increased (Table 2,3,4 & 5).This observation indicated that mortality rate varied significantly among the plants, solvents and doses.So, it is clear from the results of in vitro efficacy test that, Artemisia plants, as its crude aqueous and ethanolic extracts form, had high anthelmintic effects on eggs, larvae and adult H. contortus from goats in vitro.This was also indicated by a number of previous researchers (Akkari et al., 2014;Cala et al., 2014;Ahmed et al., 2011;Squires et al., 2011;Hart et al., 2008;Eguale et al., 2007).
From the study, it was found that A. vulgaris crude aqueous and ethanolic extracts showed significant (p<0.01) in vitro anthelmintic activity against H. contortus of goat as ascertained by egg hatching inhibition and larvae and adult worm motility inhibition.In addition, mortality was higher with the ethanolic extract treatment, whereas the worms survived for a significantly longer period in the presence of aqueous extract.This indicated that ethanolic extract has greater anthelmintic activity than the aqueous extract.

Conclusion
The present study evaluated the in vitro effects of the plant Artemisia against the eggs, larvae and adult H. contortus.Further in vitro and in vivo trials at different concentrations against different worm species and stages are required to determine a more precise anthelmintic activity of this plant species and its potential use in controlling small ruminant's gastrointestinal nematodes.

Table 1 .
Hatchability rate (%) of Haemonchus contortus eggs after treatment with crude ethanolic and aqueous extracts

Table 4 .
Mortality rate (%) of adult Haemonchus contortus worms after exposure to crude ethanolic extract treatments

Table 5 .
Mortality rate (%) of adult Haemonchus contortus worms after exposure to crude aqueous extract treatments Significant at 1% level of probability (p<0.01);NS = Not significant (p>0.05);In a column figures with same letter or without letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT)