In Vitro Combined Effects of Zanthoxylum zanthoxyloides and Newbouldia laevis Methanolic Extracts on Three Life-Cycle Stages of the Parasitic Nematode , Haemonchus contortus

| Methanolic extracts of Zanthoxylum zanthoxyloides (Fagara), Newbouldia laevis and three of their combinations (50% N. laevis -50% Z. zanthoxyloides, 25% Z. zanthoxyloides -75% N. laevis and 75% Z. zanthoxyloides 25% N. laevis) were screened in vitro for potential anti-parasitic effects against eggs, infective larvae and adults of Haemonchus contortus. Significant effects were obtained but differences were observed depending on the parasitic stage. The effects of the two plant extracts and their combinations were no dose dependent on egg hatching and infective larvae although more potent effects were usually observed with low concentrations. In contrast, dose–response relationship was found for adult worms. On eggs hatching, the combined extracts 50% N. laevis 50% Z. zanthoxyloides and 25% Z. zanthoxyloides 75% N. laevis have showed the same effectiveness like Fagara and N. laevis extracts. Combined plant extracts seemed to be more effective than individual plant extracts on larval migration at less concentrations. Extracts of Fagara and two plant associations (i.e., 50% Z. zanthoxyloides 50% N. laevis and 75% Z. zanthoxyloides 25% N. laevis) reduced adult worm motility. Overall, the combination of 50% Z. zanthoxyloides -50% N. laevis was most effective on all three stages of H. contortus. These results showed that Z. zanthoxyloides (Fagara) and N. laevis used in combined form (50%:50%) were more effective on H. contortus thus both plants association could be used as potential alternative of synthetic drugs to control parasitic infections of H. contortus in small ruminants.

many years had led to the development of resistant worm strains.Some side effects are noted and the use of disinfectants to control free stage of parasites is harmful to the environment (Wabo-Poné et al., 2011).For these reasons, alternative strategies for nematode control are needed.
In developing nations, traditional methods of controlling nematodes, used by small farmers, remain largely dependent on medicinal plants (Hounzangbé-Adoté et al., 2005a).In this regard, some ethnobotanical investigations have been undertaken everywhere in Africa and particularly in Benin on plants used by breeders to helminthes controlling.Among these plants, Zanthoxylum zanthoxyloides (Fagara), Newbouldia laevis were identified on the basis of a recent questionnaire survey in south of Benin which indicated that they were frequently used by small scale farmers against parasitic infections or to treat associated clinical signs (Hounzangbé-Adoté, 2001).Also sometime, according to Hounzangbé-Adoté (2001) leaves of both plants are used in association to helminthes controlling in this zone of Benin.In earlier studies, Hounzangbé-Adoté et al. (2005a) and(2005b) were assessed the in vitro effects of extracts from these two tropical plants on three life-cycle stages of H. contortus and Trichostrongylus colubriformis.Leaves extract of each plant was significantly reduced from each parasite, eggs hatching, larvae migration and adult worm's motility.As well, the in vivo effects of fresh and powder leaves of two plants were assessed on sheep and goat infested naturally and artificially with gastrointestinal nematodes parasites (Hounzangbe-Adoté et al., 2005c;Azando et al., 2011a;Minaflinou et al., 2015).Results obtained from these studies showed that these two plants possess anthelmintic properties and confirm their traditional uses by small farmers.However, it is important to verify if both plants used in association will be effective to helminthes controlling according to the investigations from small farmers.The current study was therefore conducted to explore the potential anti-parasitic properties of different combinations of both plants (Z.zanthoxyloides and N. laevis) from Western Africa against H. contortus.In vitro methods were employed targeting three life-cycle stages of H. contortus, i.e. the eggs, the infective larvae and the adult worms.

PreParatIon of Plant leaveS extractS
Materials screened were leaves of Z. zanthoxyloides (Fag) and leaves of N. laevis (New).Samples of each plant were collected from their natural habitat and dried indoors at room temperature.Z. zanthoxyloides (Lam.)Zepernick and Timler to Rutaceae family and N. laevis (P.Beauv.)Seemann ex Bureau to Bignoniaceae family were identified at national herbarium of Benin respectively under the numbers: AA 6301 /HNB and AA 6302 / HNB.There-after, for each plant, 50g of leaves powder were sampled and for both plants combination, respectively 37.5g/12,5g, 25g/25g and 12.5g/37,5g of powdered leaves from each plant was extracted in 500 mL of solvent for two hours at 40°C.A mixture methanol/water was used in proportion to 70:30.Following this, the solution was filtered, and the resulting filtrate was then evaporated in an oven at 47°C.Once the solvent had evaporated, the extract was obtained and dried in a drier for 5 days.

bIoloGIcal aSSaYS' ProcedureS
Extracts effects on the three main stages of the parasite cycle, i.e. the egg, the third-stage larvae (L 3 s) and the adult worms were measured using different laboratory procedures that is eggs hatching assay, inhibition of larval migration and adult motility inhibition.

effectS on H. contortus eGGS
The method was based on a modification of the egg hatch assay performed to assess anthelmintic resistance by Coles et al. (1992).Briefly, parasite eggs were freshly obtained from donor sheep experimentally infected with H. contortus.The eggs were extracted, washed repeatedly with saline and distributed in 96 multiwell plates at a concentration of 100 eggs /well in 100 µl.Following this, 100 µL of plant association extracts prepared with PBS at different concentrations (75,150,300,600,1200, and 2400 µg/mL) were added into each well containing eggs solution.A negative control (PBS) and a positive control (Thiabendazole 500, 250 and 125 µg/mL in PBS) were also included.The whole culture plate were then incubated at 27°C for 48 hours.Hatched eggs were counted under a microscope.The test was repeated five times for each plant dose.The percentage inhibition of hatching for each concentration was evaluated microscopically using the modified formula of Coles et al. (1992).

effectS on InfectIve larvae
The larval migration inhibition (LMI) bioassay was used as described by Rabel et al. (1994) in order to measure inhibiting activity against infective larvae which were obtained from stool of sheep previously infected artificially with strains of H. contortus.Droppings were collected and left to culture at room temperature for 10 days.The larvae were then extracted from the fecal mass using a Baermann apparatus, which utilizes the water tropism of larvae.After, larvae were incubated for 3 h at 20°C in PBS solutions of plant association extracts, at concentrations of 75, 150, 300 600 or 1200 µg/mL.The larvae were then washed three times in phosphate buffer (PBS) (pH 7.2, 0.15 M) and centrifuged.After the last washing, 800 µL of larvae at a concentration of 1000 L3s/mL was pipetted onto a 20 µm mesh.The sieve was inserted into a conical tube, so that it just touched the surface of the PBS contained therein.Four replicates were run at room temperature for each plant concentration.In addition, negative (larvae incubat- October 2016 | Volume 4 | Issue 4 | Page 130 ed in PBS) and positive (larvae incubated in Levamisole at concentrations of 250 µg/mL) controls were run in parallel.After 3 h, the L3s above the sieve were discarded and those which had actively migrated through the mesh into the PBS below, were counted under a microscope at magnification 20X.The percentage of LMI was calculated as:

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Where; T is the total number of L 3 deposited in the sieve and M the number of L 3 having migrated through the mesh into the PBS.

effectS on adult worMS
The purpose of this assay was to test the anthelmintic effect of the three plant extracts on adult worm motility.This test was performed according to Hounzangbé-Adoté et al. ( 2005a) and ( 2005b).Adult worms were obtained from sheep which were experimentally infected with a pure strain of H. contortus.Four weeks after infection, the sheep were euthanized.Immediately after death, the abomasum was collected, opened, briefly washed with saline and placed in a Baermann apparatus with saline at 37°C.After 2 h, the worms that had migrated into the saline were collected and quickly placed in a 48-multiwall plate at a concentration of three worms/ well.The worms were first washed for one hour in PBS with penicillin and streptomycin at a concentration of 4%.Thereafter, 1 mL of the different concentrations of plant association extracts, from 75 to 2400 µg/mL, diluted in PBS were added to the wells.Positive (Levamisole) and negative (PBS plus antibiotic) controls were included on each plate.For each treatment, the measurements were performed on six replicates per dose and per plate.The supernatant was changed every 24 h.The mobility of the adult worms was noted by careful observation under a microscope at magnification 40X after each 6h.At each observation, a motility index was calculated as the ratio between the total numbers of immobile worms/total number of worms, referring in each case to the overall number of worms present in the six replicates.

StatIStIcal analYSIS
For assays on egg hatch and larval migration, significant differences in means for the proportion of unhatched eggs and inhibition migration rates between treatments were assessed by the general linear model (GLM) procedures using package MASS (Venables and Ripley, 2002) of R software (R Core Team, 2013).Adult worm assay: for each treatment, the number of immobile worms was recorded with time and survival analyses were assessed using t test of Student.The comparison of the two plants and their combination for each dose was done using Student Newman and Keuls test (SNK) of R software.

rESultS
eGG hatch aSSaY Z. zanthoxyloides (Fagara), N. laevis extracts and their associations (50%New-50% Z. zanthoxyloides, 25% Z. zanthoxyloides -75% N. laevis and 75% Z. zanthoxyloides -25% N. laevis) have significantly (p < 0.001) reduced in vitro H. contortus eggs hatching.The reduction of eggs hatching rate have varied from 18.84 to 65% following eggs exposure to increasing concentrations (Figure 1).This inhibition was no dose-dependent (p > 0.05) but was function of the plant species tested (p < 0.001).Two plants association extracts 50% N. laevis -50% Z. zanthoxyloides and 25% Z. zanthoxyloides -75% N. laevis have showed the same effectiveness as two plants extracts (Fagara and N. laevis).The association of 75% of Fagara and 25% of N. laevis was the lowest effective (p > 0. 05).50%Fag, 25%Fag-75%New and 75%Fag-25%New) extracts was neither no dose-depend (p > 0.05) nor the plant species used (p > 0.05) (Figure 2).A significant effect on larval migration was observed for higher (1200 and 600µg/ mL) and middle (300µg/mL) concentrations extracts of each plant and each two plant associations.At weak doses, plant association extracts seemed to be more effective than two plants extracts (Figure 2).50 percent of worms were immobilized at high concentration of positive control (500 µg/mL) and less of 25 percent at middle dose (250 µg/mL) and high dose (2400 µg/mL) respectively for positive control (250 µg/ml) and Fagara extract, 6 hours after exposure.After 24 hours of incubation, total inhibition was observed in wells of positive control (500 and 250 µg/ml) and those of highest concentrations, 2400 and 1200 µg/mL for Fagara, 2400, 1200 and 600 µg/ml for 75%Fag-25%New and 50%Fag-50%New and 2400 µg/mL for 25%Fag-75%New (Table 1).A strong inhibition of H. contortus adult worms' motility was observed following exposure to each concentration of plants extracts and their combination extracts after 36 h (Table 1).The motility reduction was total for all extract doses excepted dose 1200 µg/ml of N. laevis extract (Table 1).Nevertheless, after 48 hours of exposure to different concentrations of plant extracts and their association extracts, the reduction of H. contortus motility was total.Extracts of Fagara and two plant associations 50%Fag-50%New and 75%Fag-25%New have in this assay a significant effect on the survival of adult worms of H. contortus (Table 1).

dIScuSSIOn
Zanthoxylum zanthoxyloides (Fagara) and Newbouldia laevis are present throughout tropical areas of Africa (Bekalo et al., 1996).It has been identified as plants most commonly used by farmers in Benin, as shown by a previous survey (Hounzangbé-Adoté, 2001)

AcKnOwlEdgEMEntS
Ours acknowledgments was supported by Laboratory of Ethno pharmacology and Animal Health, FSA, UAC for complementary funds through VPMAP-PAES projects.This study would not have succeeded without the role played by Traditional Healers from Benin.We finally thank the anonymous reviewers for their constructive comments on this Manuscript.

cOnFlIct OF IntErESt
There was no conflict of interest from any of Co-authors concerning this work.It was unanimously packaged.

AutHOrS' cOntrIButIOn
Irvine Yèïnou Minaflinou Sacca Sidi conducted the Biological assays in accordance with the protocol and wrote the manuscript.Géorcelin Goué Alowanou, monitored the execution of the protocol and participated in the writing of the manuscript.Pascal Abiodoun Olounladé, followed the biochemical part of the protocol, statistical analyses and participated in the correction of the manuscript.Vidjinnangni Fifamè Nadège Dedehou, monitored the execution of the protocol and participated in the writing of the manuscript.Sylvie Mawulé Hounzangbé-Adoté designed the protocol and participated in the correction of the manuscript.

of Animal Health and Production October 2016 | Volume 4 | Issue 4 | Page 132 that
. According to them the farmers usually used both plants in association for small ruminant gastro-intestinal parasites controlling.Moreover, screening based on in vitro studies using Fagara and N. laevis extracts have provided evidence indicating that different concentrations of extracts modified the viability of both adult worms and third-stage larvae of Haemonchus contortus and Trichostrongylus colubriformis and also affected egg hatching(Hounzangbe-Adote et al., 2005a, b).Recent in vivo studies of Fagara and N. laevis powder leaves realized byAzando et al. (2011a)on same parasites have confirmed both plants anthelmintic properties.However, in this time, no data was not obtained about both plants association effectiveness against nematode infections as indicated by farmers.extracts of both plants combination possessed in vitro ovicidal activity.This inhibition was function of the plant species tested and no dose-dependent.Similar observation was done on same plants and parasite in earlier study byHounzangbé-Adoté et al. (2005a)and recent study byOlounladé et al. (2011).Besides, according to Hounzangbé-Adoté et al.(2005a), the reduction in egg excretion observed in the present study suggest that plants combination extracts could affect the biology of parasitic eggs when sprayed on pastures.By itself, a reduction in egg hatching can also help to modulate the risk of parasitism by limiting the infectivity of pastures grazed by ruminants.The in vitro migration assay is a rapid and effective tool for the determination of drug effects which paralyze nematodes(D'Assonville et al., 1996).The principle of the LMI test depends on an active migration process through the sieve, the reduction in migration rate could be due either to larval mortality or to larval paralysis.These observations were remarked in the present study especially on H. contortus larvae migration.Because a significant reduction in larvae migration rate was observed especially at higher and middle concentration extracts of plant associations.The reduction of adult worms of H. contortus mobility induced by both plants combination extracts was dose dependent and function of time incubation.In this study, a high rates of immobility was recorded after 24 hours of exposure to The combination extract of 50% N. laevis -50% Z. zanthoxyloides was most effective as compared to other combinations and individual plants on at least two stages of the parasite.These results suggested that these plants could be used as alternative to synthetic drugs and accordingly as a great asset for the control of small ruminant's gastro-intestinal nematodes, particularly H. contortus.
The in vitro methods provide a means to screen rapidly for potential anthelmintic activities of the different plant extracts and to analyze the possible mechanisms involved in the interactions between active compounds and parasites.Results obtained from eggs hatching assay indicateJournal cOncluSIOnThe association extracts 50% N. laevis -50% Z. zanthoxyloides, 25% Z. zanthoxyloides -75% N. laevis and 75% Z. zanthoxyloides -25% N. laevis were effective against eggs, larvae, and adult worms of H. contortus.