Efficacy of Achyranthes aspera (L.) as a Molluscicidal Bait...

Research ArticleEfficacy of Achyranthes aspera (L) as a Molluscicidal BaitFormulation against Fresh Water Snail Biomphalaria pfeifferi

Belayhun Mandefro 12 Seid TikuMereta1 and Argaw Ambelu 1

1Department of Environmental Health Science and Technology Faculty of Public Health Jimma UniversityPO Box 378 Jimma Ethiopia2Department of Biology College of Natural and Computational Sciences Dilla University PO Box 419 Dilla Ethiopia

Correspondence should be addressed to BelayhunMandefro belayhunmandefrogmailcom

Received 11 April 2018 Accepted 3 June 2018 Published 27 June 2018

Academic Editor Samra Bashir

Copyright copy 2018 Belayhun Mandefro et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Molluscicidal plant products have interesting attributes of environmental friendliness and accessibility to local communitiesTheirbait formulations are more economical and target specific as they are applied only to the snail-infested sections of the waterhabitat Their active ingestion by target snails could also increase gastric concentrations and enhance effectiveness This studyaimed to evaluate the comparative effectiveness ofAchyranthes aspera (A aspera) leaf hydroethanolic extract in bait and immersionapplications Serial dilutions of the extract inwater for immersion and in snail food pellets for bait test were set AdultBiomphalariapfeifferi snails exposed to the bioassays for 24 hours and data were analyzed using probit model The plant showed molluscicidalactivity in both methods The respective LC

50and LC

90doses were 2037 and 4684 ppm in the immersion and 310 and 1108

ppm in the bait The more efficient bait method reduced the LC50

by 657 and the LC90

by 423 times This finding providesa strong foundation for the molluscicidal potential of A aspera It is applicable and possibly more effective if formulated intothose commercially available snail food pellets or flakes However selection and optimization of suitable baits is a crucial step formaximum output

1 Background

Schistosomiasis control can be more successful whenchemotherapy and snail control activities are integrated[1 2] Recently molluscicidal plants are drawing moreand more attention for their environmental friendlinessaccessibility and cost effectiveness They are especiallysuitable for community based snail control activities inplaces where schistosomiasis transmission is more focal[3] So far several molluscicidal plants have been screenedworldwide Carica papaya (C papaya) Terminalia catappa(T catappa) [4] and Solanum species [5] are just someof them Glinus lotoides (G lotoides) [6] and Puerariapeduncularis (P peduncularis ) [7] are also among therecently discovered ones

The relatively low efficacy of many molluscicidal plantsas compared to niclosamide is a hindrance to their practicalapplicability [8] Parallel to the search for noble molluscicides

of higher efficacy and safety adoption of efficient applicationmechanisms could play a decisive role in enhancing theireffectiveness In principle such molluscicides should beapplied in dose as low as possible for environmental naturalresource conservation and economic reasons [9 10] In theusual immersion method the molluscicide is added to thewater habitat [11 12] and is needed in large amount to titratethe entire water body Alternatively molluscicidal baits areformulated irrespective of the water volume and are appliedonly to the snail-infested sections requiring only a very smallamount of active ingredients Baits use as carriers of themolluscicide and attractants which increase palatability ofthe substance by target snails [13ndash15] Such baits have beenpracticed against freshwater snail Lymnaea acuminata bysome researchers [16ndash18]

Achyranthes aspera (A aspera) is a perineal herb in thefamily Amaranthaceae The plant grows in many parts ofEthiopia on roadsides on wastelands near hedges and under

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 2718585 7 pageshttpsdoiorg10115520182718585

2 Evidence-Based Complementary and Alternative Medicine

eucalyptus tree plantations It is a known medicinal plant inEthiopian folklore used for placental retention postpartumbleeding skin eruptions and wound dressing [19 20] Thisstudy is aimed to evaluate the comparative efficacy of Aaspera leaf hydroethanolic extract against Biomphalaria pfeif-feri (B pfeifferi) snails in molluscicidal bait and immersionapplications in the laboratory condition

2 Material and Methods

21 Plant Material Collection and Processing The plant wasselected for this study based on the researchersrsquo previousscreening tests Mature green leaves were collected in Octo-ber 2016 from a natural habitat located at 9∘431015840455910158401015840 N39∘73101584027110158401015840 E central Ethiopia It was dried in the shadeand ground to fine powder of 200 120583m mesh size as inAdenusi and Odaibo [21] It was stored in plastic bags in thelaboratory of Environmental Health Science and TechnologyJimma University Ethiopia Experts in Addis Ababa Univer-sity Herbarium have identified and authenticated the plantspecies Voucher specimen with specimen number MB1 iskept in the herbarium

22 Extract Preparation Considering the relative polarity ofthe presumedmolluscicidal phytochemicals such as saponins[1 22] hydroethanol is selected as an efficient extractionsolvent In addition hydroethanol renders more extractyield than absolute ethanol [22 23] Ethanol is also saferand less toxic than methanol acetone and other organicsolvents Extraction was done with slight modification ofthe procedure in Ndamukong and colleagues [24] Exactly100 g plant powder was rinsed in 1000 ml of 80 ethanolin a flat-bottomed airtight flask and shook on an orbitalshaker for 24 hours at 125 rpm at room temperature Afterfiltration with Whatman filter paper (110 mm thickness CATNo 1540 110) the filtrate evaporated from a wide mouthbeaker placed in a water bath at 40∘C The resulting blue-black sticky amorphous matrix was stored in a dry cleancontainer

23 Phytochemical Analysis Qualitative phytochemicalscreening tests were done on the extract based on standardprocedures described in Akinyemi et al [25] Sasidharanet al [26] and Mungenge et al [27] Reducing sugarswere identified by Fehlingrsquos test Biuret test was used forproteins froth test for saponins 5 ferric chloride fortotal phenolics and 10 ferric chloride and gelatin test fortannins Alkaline test and concentrated sulphuric acid inammonia were used for flavonoid test In addition Wagnerrsquostest for alkaloids Salkowski reaction for terpenoids andLiebermann-Burchard test for steroids were applied

24 Snail Food Preparation We used lettuce (Lactuca sativa)leaves for snail food About 2 kg of young leaves separatedfrom the stems and midribs was partially cooked in boilingtap water for 2 minutes The par-boiled leaves were driedunder the shade on a clean plastic sheet It was ground to afine powder of 250 120583mmesh size and stored in airtight plasticbags

25 Snail Collection and Maintenance We collected adultsnails of B pfeifferi from streamhabitats located at 7∘411015840153910158401015840N 36∘501015840523210158401015840 E in Jimma Town Public Prison agriculturalfield southwestern Ethiopia Snails were transported to thelaboratory in clean plastic buckets half-filled with water fromthe streams We acclimatized them in the laboratory for oneweek in 3 clean large plastic buckets filled with 10 l aged watereach containing 200 snails at room temperature under 12 hlight and 12 h dark photoperiods Snails were fed with thepreviously prepared lettuce leaf powder by spreading about025 g powder into each bucket in every 24 hours and waterwas replaced in every 3 daysThe snailswere ethically handledthroughout the experiment according to the principles andguidelines of animal welfare in scientific researches [28 29]They were not subjected to cercaria shedding before or afterthe experiment However an independent and contemporarystudy indicated a 285 snail infection rate (Bedewi et alunpublished)

26 Molluscicidal Test Assay in Immersion Method Themolluscicidal test assay was established based on standardprocedures adopted for immersion type of test [30] Stocksolution of 1000 ppm was prepared by dissolving 05 g plantextract in 500 ml aged water From this 100 ml dilutionsof 313 625 125 25 50 and 100 ppm were prepared inclean beakers Ten adult B pfeifferi snails shell diameter 95-106 mm were exposed to each dilution without food for24 hours at room temperature The test was performed inthree replicates Positive control was prepared from 15 ppmniclosamide and negative control from only aged water After24 hours the snails in their respective groups were removedfrom the solutions washed with aged water in a childcaremanner and transferred to new beakers with aged water andfood for another 24 hours Finally dead and alive snails werecounted through careful inspection Snails were considereddead if they lose sense when the foot is pocked with needleor if they remain retracted in to the shell or else the foot isdiscolored [6 30]

27 Molluscicidal Test Assay in Bait Method Molluscicidalbaits in the form of pellets were prepared based on themethods in Laskowski and Hopkin [31] and Srivastava et al[18] Different masses of the extract 0024 0047 0094 01880375 0750 and 1500 mg dissolve in 10 ml distilled waterTo each of these solutions a 5 g lettuce powder was added tomake a 15 g homogenized pellet Hence the correspondingconcentrations of the extract in the pellets were 157 313625 125 25 and 50 ppm Each pellet was evenly pasted onthe inside base of a clean Petri dish (95 mm diameter) bypressing with smooth flat wooden applicator Ten adult Bpfeifferi snails of 95-108 mm shell diameter were releasedin to each Petri dish for 24 hours Wet cotton was put overthe snails to maintain wet environment as adapted fromLaskowski and Hopkin [31] The test was carried out in fourreplicates with positive control made from pellet consistingof 15 ppm niclosamide (Bayer and Pro-Serv Inc Germany)and negative control pellet from only lettuce powder After24 hours the snails from each bait were collected carefullywashed andmaintained in clean beakers with aged water and

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Results of phytochemical screening tests on A aspera leaf hydroethanolic extract Here their relative abundances are expressed as+++ for more abundant ++ for moderately abundant + for trace and ndash for none

Phytoconstituents Screening tests Observationreducing sugars Fehlingrsquos test +proteins Biuretrsquos test -saponins frothfoam test +++total phenolics 5 FeCl

3test ++

tannins 10 FeCl3test -

gelatin test -

flavonoids reaction with H2SO4in ammonia +++

alkaline test (KOH) -alkaloids Wagnerrsquos test ++terpenoids Salkowski reaction +steroids Liebermann-Burchard reaction +

Table 2Mortality data showing the total number of exposed and dead B pfeifferi snails in each concentrationofA aspera leaf hydroethanolicextract during immersion and bait tests

Immersion test Bait testconcentration (ppm) exposed dead concentration (ppm) exposed dead000 30 0 000 40 2313 30 0 157 40 7625 30 2 313 40 231250 30 7 625 40 322500 30 15 1250 40 375000 30 29 2500 40 3810000 30 30 5000 40 40

food for another 24 hours Finally live and dead snails in eachtreatment group were counted with careful inspection Snaildeath was confirmed by loss of sense when pocked with aneedle or by discolored mantle

28 Data Analysis The mortality data were analyzed usingprobit regression model in IBM SPSS program version 23to determine the LC

50and LC

90doses with their respective

upper and lower limits [32 33] These are the principalendpoints considered as a measure of molluscicidal efficacyIn addition the nature of correlations existing betweenconcentrations and mortality rates were assessed from theslope function

3 Results

31 Phytochemical Analysis of the LeafHydroethanolic ExtractThe phytochemical screening test results (Table 1) revealedthat the extract consists of a number of bioactive secondarymetabolites Saponins and flavonoids were found in relativeabundance but tannins and proteins were not detected in thisparticular test

32 Molluscicidal Activity In the immersion test snailsexhibited irritative behavior and try to escape from the solu-tions by crawling up beyond the water level They producedmore mucus Bleeding was observed later in the course of

the experiment They started to show signs of paralysis ordeath after the first six hours of exposure When snails wereexposed to the molluscicidal baits they come out of theirshells and move about over the pellet in the first hour Thenthey gradually became inactive reluctant tomove and startedretracting to their shells During the rehabilitation periodsurvivors come out of their shells and resumed activity

Snail mortalities in the two tests are presented in Table 2The record clearly showed that mortality rates increase withincreasing concentrations of the extract

The resulting probit analysis (Table 3) shows that thecalculated 1205942 values are less than the tabular valuesThis indi-cates that the data are qualified for Pearsonrsquos goodness of fitIn addition the positive slope indicates positive correlationsbetween mortality rates and concentrations

4 Discussion

The findings of this study revealed A aspera has aconcentration-dependent molluscicidal effect against adultB pfeifferi snails in both immersion and bait applicationsHowever the two methods exhibited different efficaciesThe LC

50concentration in the bait is smaller than that

in the immersion test (Table 3) This indicates the plant ismore efficacious when applied as molluscicidal bait than itsimmersion form


Table 3The LC50and LC

90doses (with confidence limits) of A aspera leaf hydroethanolic extract against B pfeifferi snails in immersion and

bait tests

Test method Effective doses Confidence limits 1205942 (95 CL) Slope

Immersion LC50= 2037 1697-2457 4158 3544

LC90= 4684 3686-6712

Bait LC50= 310 129-516 4760 2319

LC90= 1108 704-1700

Previous studies have shown that the aqueous extractof A aspera is effective against B pfeifferi snails with 724ppm LC

50and 965 ppm LC

90[34] In the current study the

hydroethanolic extract exhibited a higher potency (2037 ppmLC50and 4684 ppmLC

90) by the same immersion technique

Similar to many other studies [35] the alcoholic extract ismore potent than the aqueous one Ethanol consists of bothpolar and nonpolar terminals at its opposite endsTherefore itis capable of dissolving and extracting multitudes of organiccompounds with a range of polarity

Phytochemical analysis of this particular extract indi-cated presence of saponins flavonoids alkaloids and totalphenolics Thus the molluscicidal property of this plant isdue to the availability of these bioactive phytoconstituents[1 36] Saponins are importantmolluscicidal compounds thatdisrupt cellular permeability and osmoregulatory functionsby interacting with membrane sterols of cells [5] Alkaloidsand terpenoids mainly act as acetylcholinesterase inhibitorsresulting in neurotoxicity in snails [30 37]

There are studies regarding the toxicological and pharma-cological aspects of A aspera Many researches investigatedno significant mammalian toxicity of the plant [38 39]Rather its antioxidant and anti-inflammatory properties dueto the presence of saponins alkaloids and oleanolic acid[40 41] are well known On the other hand antifertility andspermicidal properties [42 43] as well as larvicidal propertyagainst Ae aegypti [44] are indicated

Several plants have exhibited molluscicidal activityagainst B pfeifferi snails and related species In the currentstudy the LC

50from hydroethanolic extract of A aspera leaf

is 2037 ppm It is proved to bemore potent thanCymbopogoncitratus where the LC

50is 15947ppm [45] It is also more

potent than G lotoides aqueous and ethyl acetate extracts inKiros et al [6] as well as Entada leptostachya and Azadirachtaindicamethanol extracts in Michael and colleagues [46]

The current study also showed a higher molluscicidalefficacy ofA aspera in the bait application as compared to theordinary immersion method (Table 3) The bait applicationreduced the LC

50and LC

90lethal concentrations by about

66 and 42 times respectively The enhancement on the LC50

is greater than that on the LC90 Generally greater efficacy

enhancement was achieved in the sublethal doses than in thehigher doses (Figure 1) According to previous studies highermolluscicide titers could alter the natural taste and attractantquality of the snail food Snails may hesitate to consume toomuch of it hence reducing its palatability and effectiveness

In line with this result some studies have reportedenhanced molluscicidal effectiveness in bait formulations [1617] Nevertheless bait effectiveness is a function of several

Figure 1 Comparative molluscicidal efficacy of A aspera leafhydroethanolic extract in bait and immersion tests againstB pfeifferiadult snails (broken line represents the bait test solid line representsthe immersion test)

factors such as palatability attractant property snail lifestages andmany others [14 17 47]The present study impliesthat alternative molluscicide applications such as baits withappropriate snail foods and attractants can be one possiblemechanism to enhance the efficacy

In immersion application the molluscicide action ismainly by contact poisoning of the epithelial tissues inthe head-foot region Different studies reported closing ofoperculum increased mucus secretion and retracting intothe shell during immersion [48] Such protective behaviorscan prevent free imbibition of molluscicidal compounds intothe inner body cavities On the other hand molluscicidalbaits act as stomachpoisons [49]Hence damage to the gastricepithelium is the main mechanism of action [14 50 51]Increased efficacy in molluscicidal bait application can alsobe associated with other related factors If snails avidly ingestthe bioactive compounds with the food they may accumulatein the gut to a higher local concentration at least temporarilybefore excretion As the local concentration increases greaterdamage is certainly expected On the other hand the gastricepithelium provides a larger area of poison contact than thecephalopodal surface It may also facilitate the compoundsrsquodispersal to hepatic tissues and other vital organs


Molluscicidal bait application in the control of aquaticsnails has additional advantages It prevents chemicals fromdirect dispersal in to the aquatic environment Attractants inthe bait lure target snails so that they ingest the molluscicideactively On the other hand since the bait is applied onlyto the snail-infested section of the water habitat it is moreeconomical and target specific [16 52]

5 Conclusion

Molluscicide application in the field needs several consid-erations It should be applied in lowest possible dose tominimize interference with the physicochemical propertiesof the aquatic habitats and adverse effects on nontargetorganisms It should also be economical for sustainable use ofthose valuable medicinal plantsTherefore selection of plantswith better molluscicidal efficacy and safety is mandatoryBesides modified application techniques that enhance theireffectiveness are equally important

This study confirms the molluscicidal activity of Aaspera from hydroethanolic extract of the leaf part in bothimmersion and bait application techniques The findingsprovide a strong foundation to the molluscicidal potentialof this plant for community based snail control activitiesFurthermore an appreciably higher efficacy resulted from thebait test indicating possibility of its application by formulatinginto those commercially available snail food pellets or flakesThe research further indicated that bait applications couldbe one possible mechanism to enhance molluscicidal efficacyof many claimed plants as long as appropriate foods andattractants are used

Data Availability

All data required for the analysis and conclusion are includedin this article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Belayhun Mandefro conceived the study idea carried outthe experiment wrote the manuscript draft and preparedthe final manuscript Seid TikuMereta supervised the exper-iment process organized literature search and backgroundinformation and reviewed the final manuscript ArgawAmbelu modified the experimental design analyzed andinterpreted data and reviewed the final manuscript

Acknowledgments

The authors thankfully acknowledge Jimma University andDilla University Ethiopia for their financial support Theygot professional advices regarding the statistical model fromMr Kibralem and the late Mr Girma Tefera (Jimma Univer-sity Ethiopia) Mr Yifrashewa Mengesha (Jimma UniversityEthiopia) has helped in snail collection andmaintenanceThe

authors are grateful to all of them This work was supportedby Jimma University and Dilla University Ethiopia

References

[1] A A Pereira Filho C R C Franca D D S Oliveira R J DA Mendes J D R S Goncalves and I G Rosa ldquoEvaluationof the molluscicidal potential of hydroalcoholic extracts ofJatropha gossypiifolia Linnaeus 1753 on Biomphalaria glabrata(Say 1818)rdquo Revista do Instituto de Medicina Tropical de SaoPaulo vol 56 no 6 pp 505ndash510 2014

[2] P M Z Coelho and R L Caldeira ldquoCritical analysis ofmolluscicide application in schistosomiasis control programs inBrazilrdquo Infectious Diseases of Poverty vol 5 no 1 article no 572016

[3] D Rollinson S Knopp S Levitz et al ldquoTime to set the agendafor schistosomiasis eliminationrdquoActa Tropica vol 128 no 2 pp423ndash440 2013

[4] V O Adetunji and O T Salawu ldquoEfficacy of ethanolic leafextracts of Carica papaya and Terminalia catappa as mollusci-cides against the snail intermediate hosts of schistosomiasisrdquoJournal of Medicinal Plants Research vol 4 no 22 pp 2348ndash2352 2010

[5] G El Sherbini R Zayed and E El Sherbini ldquoMolluscicidalActivity of Some Solanum Species Extracts against the SnailBiomphalaria alexandrinardquo Planta Medica vol 77 no 12 2011

[6] G Kiros B Erko M Giday and Y Mekonnen ldquoLaboratoryassessment of molluscicidal and cercariacidal effects of Glinuslotoides fruitsrdquo BMC Research Notes vol 7 article 220 2014

[7] C Yang M Zhang B Lei et al ldquoActive saponins from root ofPueraria peduncularis (Grah ex Benth) Benth and their mol-luscicidal effects on Pomacea canaliculatardquo Pest ManagementScience vol 73 no 6 pp 1143ndash1147 2017

[8] FHanif andDK Singh ldquoBinary combination ofCarica papayaAreca catechu and Myristica fragrans with piperonyl butoxideMGK-264 against freshwater snail Lymnaea acuminatardquo Tropi-cal Life Sciences Research vol 24 no 2 pp 1ndash11 2013

[9] J Shahi and A Singh ldquoEffect of bioactive compounds extractedfrom euphorbious plants on hematological and biochemicalparameters of Channa punctatusrdquoRevista do Instituto deMedic-ina Tropical de Sao Paulo vol 53 no 5 pp 259ndash263 2011

[10] R P Yadav and A Singh ldquoEfficacy of euphorbia hirta latex asplant derived molluscicides against freshwater snailsrdquo Revistado Instituto de Medicina Tropical de Sao Paulo vol 53 no 2 pp101ndash106 2011

[11] J Xia Y Yuan X Xu et al ldquoEvaluating the effect of a novelmolluscicide in the endemic schistosomiasis japonica area ofChinardquo International Journal of Environmental Research andPublic Health vol 11 no 10 pp 10406ndash10418 2014

[12] C H King D Bertsch and M Knight ldquoHistorical PerspectiveSnail Control to Prevent Schistosomiasisrdquo PLOS NeglectedTropical Diseases vol 9 no 4 p e0003657 2015

[13] C A Edwards N Q Arancon M Vasko-Bennett B Littleand A Askar ldquoThe relative toxicity of metaldehyde and ironphosphate-based molluscicides to earthwormsrdquo Crop Protec-tion vol 28 no 4 pp 289ndash294 2009

[14] T R Smith J White-Mciean K Dickens A C Howe andA Fox ldquoEfficacy of four molluscicides against the giantAfrican Snail Lissachatina fulica (Gastropoda PulmonataAchitinidae)rdquo Florida Entomologist vol 96 no 2 pp 396ndash4022013


[15] D N Cardoso M J G Santos A M V M Soares and SLoureiro ldquoMolluscicide baits impair the life traits of Folsomiacandida (Collembola) Possible hazard to the population leveland soil functionrdquo Chemosphere vol 132 pp 1ndash7 2015

[16] F Tiwari ldquoBait formulation toxicity of plant derived mollusci-cides in attractant food pellets against vector snail Lymnaeaacuminatardquo World Journal of Zoology vol 7 no 1 pp 54ndash592012

[17] P Kumar V K Singh and D K Singh ldquoReproduction ofLymnaea acuminata fed to bait containing binary combinationof amino acids with mollusciciderdquo Journal of Biology and EarthSciences vol 3 no 1 pp B65ndashB71 2013

[18] Singh V K Singh and A K Srivastava ldquoAbiotic factors andanti-reproductive action of bait containing eugenol againstLymnaea acuminatardquo The Journal of Biological Sciences vol 2no 4 pp 76ndash85 2013

[19] S Kalayou M Haileselassie G Gebre-egziabher et al ldquoIn-vitro antimicrobial activity screening of some ethnoveterinarymedicinal plants traditionally used against mastitis wound andgastrointestinal tract complication in Tigray Region EthiopiardquoAsian Pacific Journal of Tropical Biomedicine vol 2 no 7 pp516ndash522 2012

[20] B Tadesse G Mulugeta G Fikadu and A Sultan ldquoSurvey onethno-veterinary medicinal plants in selected Woredas of eastWollega zone western Ethiopiardquo Journal of Biology Agricultureand Healthcare vol 4 no 17 pp 97ndash105 2014

[21] A A Adenusi and A B Odaibo ldquoEffects of varying con-centrations of the crude aqueous and ethanolic extracts ofDalbergia sissoo plant parts on Biomphalaria pfeifferi eggmassesrdquo African Journal of Traditional Complementary andAlternative Medicines vol 6 no 2 pp 139ndash149 2009

[22] J Kwon G Lee J M Belanger and J R Jocelyn Pare ldquoEffect ofethanol concentration on the efficiency of extraction of ginsengsaponins when using a microwave-assisted process (MAPtm)rdquoInternational Journal of Food Science amp Technology vol 38 no5 pp 615ndash622 2003

[23] B Sultana F Anwar and M Ashraf ldquoEffect of extraction sol-venttechnique on the antioxidant activity of selectedmedicinalplant extractsrdquoMolecules vol 14 no 6 pp 2167ndash2180 2009

[24] K J N Ndamukong N N Ntonifor J Mbuh A F Atemnkengand M T Akam ldquoMolluscicidal activity of some cameroonianplants on Bulinus speciesrdquo East AfricanMedical Journal vol 83no 3 pp 102ndash109 2006

[25] K O Akinyemi O Oladapo C E Okwara C C Ibe andK A Fasure ldquoScreening of crude extracts of six medicinalplants used in South-West Nigerian unorthodox medicine foranti-methicillin resistant Staphylococcus aureus activityrdquo BMCComplementary and Alternative Medicine vol 5 article 6 2005

[26] S Sasidharan Y ChenD Saravanan K Sundram and L LathaldquoExtraction Isolation AndCharacterizationOf Bioactive Com-pounds From Plantsrsquo Extractsrdquo African Journal of TraditionalComplementary and Alternative Medicines vol 8 no 1 2010

[27] C Mungenge C Zimudzi M Zimba and T NhiwatiwaldquoPhytochemical screening cytotoxicity and insecticidal activ-ity of the fish poison plant Synaptolepis alternifolia Oliv(Thymelaeaceae)rdquo Journal of Porphyrins and Phthalocyaninesvol 2 no 5 pp 15ndash19 2014

[28] American Psychological Association (APA) Guidelines forethical conduct in the care and use of nonhuman animals inresearch Committee on Animal Research and Ethics (CARE)Washington DC 2018

[29] National Institutes of Health (NIH) PHS policy on humanecare and use of laboratory animals National Institutes of Health(NIH) Bethesda Maryland 2015

[30] M M Salama E E Taher and M M El-Bahy ldquoMolluscicidaland mosquitocidal activities of the essential oils of thymuscapitatus Hoff ET link and marrubium vulgare Lrdquo Revista doInstituto de Medicina Tropical de Sao Paulo vol 54 no 5 pp281ndash286 2012

[31] R Laskowski and S P Hopkin ldquoEffect of Zn Cu Pb andCd on fitness in snails (Helix aspersa)rdquo Ecotoxicology andEnvironmental Safety vol 34 no 1 pp 59ndash69 1996

[32] C Weber ldquoAcute Toxicity Data Analysisrdquo in Methods formeasuring the acute toxicity of effluents and receiving watersto freshwater and marine organisms pp 66ndash77 EnvironmentalMonitoring Systems Laboratory Office of Research and Devel-opment US Environmental Protection Agency CincinnatiOhio 4th edition 1991

[33] G Otieno G A Waititu and D Salifu ldquoGeneralized Estimat-ing Equations for Repeated Measures Logistic Regression inMosquitoDose-ResponserdquoOpen Journal of Statistics vol 03 no05 pp 293ndash298 2013

[34] BMandefro S T Mereta Y Tariku and A Ambelu ldquoMollusci-cidal effect of Achyranthes aspera L (Amaranthaceae) aqueousextract on adult snails of Biomphalaria pfeifferi and Lymnaeanatalensisrdquo Infectious Diseases of Poverty vol 6 no 1 2017

[35] A El-Din K El-Sayed and M Mahmoud ldquoEffect of ethanolicextract of Dalbergia sissoo plant parts on Biomphalaria alexan-drina snail the intermediate host of Schistosoma mansonirdquoJournal of Evolutionary Biology Research vol 3 no 7 pp 95ndash100 2011

[36] H Hammami R Mezghani-Jarraya M Damak and A AyadildquoMolluscicidal activity of various solvent extracts from SolanumNigrum VarVillosum L Aerial parts against Galba TruncatulardquoParasite vol 18 no 1 pp 63ndash70 2011

[37] S K Singh R P Yadav andA Singh ldquoMolluscicides from somecommon medicinal plants of eastern Uttar Pradesh IndiardquoJournal of Applied Toxicology vol 30 no 1 pp 1ndash7 2010

[38] C Reddy and A Kamble ldquoToxicity study of Achyranthusasperardquo International letters of natural sciences vol 14 pp 85ndash96 2014

[39] A Habtamu and Y Mekonnen ldquoEvaluation of the antibacterialactivities of leaf extracts of Achyranthus asperardquoAfrican Journalof Bacteriology Research vol 9 no 2 p 14 2017

[40] C Priya G Kumar L Karthik and K Bhaskara Rao ldquoPhy-tochemical composition and in vitro antioxidant activity ofAchyranthes aspera Linn (Amaranthaceae) leaf extractsrdquo Jour-nal of Agricultural Science and Technology vol 8 no 1 pp 143ndash156 2012

[41] P S Kothavade V D Bulani D M Nagmoti P S DeshpandeN B Gawali and A R Juvekar ldquoTherapeutic effect of saponinrich fraction of Achyranthes aspera Linn on adjuvant-inducedarthritis in sprague-dawley ratsrdquo Autoimmune Diseases vol2015 2015

[42] A Dey ldquoAchyranthes aspera L phytochemical and pharmaco-logical aspectsrdquo International Journal of Pharmaceutical SciencesReview and Research vol 9 no 2 2011

[43] R B Desi Reddy P Narisi Reddy N Prathibha T MadhuMounica A Phanikumar andMRavindra ldquoPharmacognosticpreliminary phyto chemical and pharmacological studies on theroots of achyranthes asperardquo International Journal of Pharma-cognosy and Phytochemical Research vol 4 no 3 pp 139ndash1412012


[44] A Sharma S Kumar and P Tripathi ldquoImpact of Achyranthesaspera leaf and stem extracts on the survival morphology andbehaviour of an Indian strain of dengue vector Aedes aegypti l(diptera culicidae)rdquo Journal of Mosquito Research vol 5 no 7pp 1ndash9 2015

[45] B Otarigho and O A Morenikeji ldquoMolluscicidal effects ofaqueous and ethanolic extracts of Lemongrass (Cymbopogoncitratus) leaf against the different developmental stages ofBiomphalaria pfeifferirdquo New York Science Journal vol 5 no 8pp 70ndash77 2012

[46] E Michael D Yole M Musila H Kutima and P KareruldquoAssessment of molluscicidal cercericidal and miracicidalactivities of crude extracts of Azadirachta indica and Entadaleptostachyardquo Journal of Biology Agriculture andHealthcare vol3 no 5 pp 11ndash17 2013

[47] N B Bourne GW Jones and I D Bowen ldquoFeeding behaviourand mortality of the slug Deroceras reticulatum in relationto control with molluscicidal baits containing various combi-nations of metaldehyde with methiocarbrdquo Annals of AppliedBiology vol 117 no 2 pp 455ndash468 1990

[48] S Kamble and N Kamble ldquoBehavioural changes in freshwatersnail Bellamya bengalensis due to acute toxicity of coppersulphate and Acacia sinuatardquo International Journal of Environ-mental Science and Technology vol 3 no 3 pp 1090ndash1104 2014

[49] C Salvio A J Faberi A N Lopez P L Manetti and N LClemente ldquoThe efficacy of three metaldehyde pellets marketedin Argentina on the control of Deroceras reticulatum (Muller)(Pulmonata Stylommatophora)rdquo Spanish Journal of Agricul-tural Research vol 6 no 1 pp 70ndash77 2008

[50] I E Ebenso ldquoMolluscicidal effects of neem (Azadirachtaindica) extracts on edible tropical land snailsrdquo Pest ManagementScience vol 60 no 2 pp 178ndash182 2004

[51] D Gonzalez-Cruz and R San Martın ldquoMolluscicidal effects ofsaponin-rich plant extracts on the grey field slugrdquo Ciencia eInvestigacion Agraria vol 40 no 2 pp 341ndash349 2013

[52] P Kumar V K Singh and D K Singh ldquoAttractant foodpellets containing molluscicides against the fresh water snailIndoplanorbis exustusrdquoGlobal Veterinaria vol 8 no 2 pp 105ndash110 2012

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eucalyptus tree plantations It is a known medicinal plant inEthiopian folklore used for placental retention postpartumbleeding skin eruptions and wound dressing [19 20] Thisstudy is aimed to evaluate the comparative efficacy of Aaspera leaf hydroethanolic extract against Biomphalaria pfeif-feri (B pfeifferi) snails in molluscicidal bait and immersionapplications in the laboratory condition

2 Material and Methods

21 Plant Material Collection and Processing The plant wasselected for this study based on the researchersrsquo previousscreening tests Mature green leaves were collected in Octo-ber 2016 from a natural habitat located at 9∘431015840455910158401015840 N39∘73101584027110158401015840 E central Ethiopia It was dried in the shadeand ground to fine powder of 200 120583m mesh size as inAdenusi and Odaibo [21] It was stored in plastic bags in thelaboratory of Environmental Health Science and TechnologyJimma University Ethiopia Experts in Addis Ababa Univer-sity Herbarium have identified and authenticated the plantspecies Voucher specimen with specimen number MB1 iskept in the herbarium

22 Extract Preparation Considering the relative polarity ofthe presumedmolluscicidal phytochemicals such as saponins[1 22] hydroethanol is selected as an efficient extractionsolvent In addition hydroethanol renders more extractyield than absolute ethanol [22 23] Ethanol is also saferand less toxic than methanol acetone and other organicsolvents Extraction was done with slight modification ofthe procedure in Ndamukong and colleagues [24] Exactly100 g plant powder was rinsed in 1000 ml of 80 ethanolin a flat-bottomed airtight flask and shook on an orbitalshaker for 24 hours at 125 rpm at room temperature Afterfiltration with Whatman filter paper (110 mm thickness CATNo 1540 110) the filtrate evaporated from a wide mouthbeaker placed in a water bath at 40∘C The resulting blue-black sticky amorphous matrix was stored in a dry cleancontainer

23 Phytochemical Analysis Qualitative phytochemicalscreening tests were done on the extract based on standardprocedures described in Akinyemi et al [25] Sasidharanet al [26] and Mungenge et al [27] Reducing sugarswere identified by Fehlingrsquos test Biuret test was used forproteins froth test for saponins 5 ferric chloride fortotal phenolics and 10 ferric chloride and gelatin test fortannins Alkaline test and concentrated sulphuric acid inammonia were used for flavonoid test In addition Wagnerrsquostest for alkaloids Salkowski reaction for terpenoids andLiebermann-Burchard test for steroids were applied

24 Snail Food Preparation We used lettuce (Lactuca sativa)leaves for snail food About 2 kg of young leaves separatedfrom the stems and midribs was partially cooked in boilingtap water for 2 minutes The par-boiled leaves were driedunder the shade on a clean plastic sheet It was ground to afine powder of 250 120583mmesh size and stored in airtight plasticbags

25 Snail Collection and Maintenance We collected adultsnails of B pfeifferi from streamhabitats located at 7∘411015840153910158401015840N 36∘501015840523210158401015840 E in Jimma Town Public Prison agriculturalfield southwestern Ethiopia Snails were transported to thelaboratory in clean plastic buckets half-filled with water fromthe streams We acclimatized them in the laboratory for oneweek in 3 clean large plastic buckets filled with 10 l aged watereach containing 200 snails at room temperature under 12 hlight and 12 h dark photoperiods Snails were fed with thepreviously prepared lettuce leaf powder by spreading about025 g powder into each bucket in every 24 hours and waterwas replaced in every 3 daysThe snailswere ethically handledthroughout the experiment according to the principles andguidelines of animal welfare in scientific researches [28 29]They were not subjected to cercaria shedding before or afterthe experiment However an independent and contemporarystudy indicated a 285 snail infection rate (Bedewi et alunpublished)

26 Molluscicidal Test Assay in Immersion Method Themolluscicidal test assay was established based on standardprocedures adopted for immersion type of test [30] Stocksolution of 1000 ppm was prepared by dissolving 05 g plantextract in 500 ml aged water From this 100 ml dilutionsof 313 625 125 25 50 and 100 ppm were prepared inclean beakers Ten adult B pfeifferi snails shell diameter 95-106 mm were exposed to each dilution without food for24 hours at room temperature The test was performed inthree replicates Positive control was prepared from 15 ppmniclosamide and negative control from only aged water After24 hours the snails in their respective groups were removedfrom the solutions washed with aged water in a childcaremanner and transferred to new beakers with aged water andfood for another 24 hours Finally dead and alive snails werecounted through careful inspection Snails were considereddead if they lose sense when the foot is pocked with needleor if they remain retracted in to the shell or else the foot isdiscolored [6 30]

27 Molluscicidal Test Assay in Bait Method Molluscicidalbaits in the form of pellets were prepared based on themethods in Laskowski and Hopkin [31] and Srivastava et al[18] Different masses of the extract 0024 0047 0094 01880375 0750 and 1500 mg dissolve in 10 ml distilled waterTo each of these solutions a 5 g lettuce powder was added tomake a 15 g homogenized pellet Hence the correspondingconcentrations of the extract in the pellets were 157 313625 125 25 and 50 ppm Each pellet was evenly pasted onthe inside base of a clean Petri dish (95 mm diameter) bypressing with smooth flat wooden applicator Ten adult Bpfeifferi snails of 95-108 mm shell diameter were releasedin to each Petri dish for 24 hours Wet cotton was put overthe snails to maintain wet environment as adapted fromLaskowski and Hopkin [31] The test was carried out in fourreplicates with positive control made from pellet consistingof 15 ppm niclosamide (Bayer and Pro-Serv Inc Germany)and negative control pellet from only lettuce powder After24 hours the snails from each bait were collected carefullywashed andmaintained in clean beakers with aged water and




3test ++


gelatin test -







50and LC



3 Results






4 Discussion







bait tests


Immersion LC50= 2037 1697-2457 4158 3544

LC90= 4684 3686-6712

Bait LC50= 310 129-516 4760 2319

LC90= 1108 704-1700


50and 965 ppm LC













50and LC











5 Conclusion



Data Availability






Acknowledgments



References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















3test ++


gelatin test -







50and LC



3 Results






4 Discussion







bait tests


Immersion LC50= 2037 1697-2457 4158 3544

LC90= 4684 3686-6712

Bait LC50= 310 129-516 4760 2319

LC90= 1108 704-1700


50and 965 ppm LC













50and LC











5 Conclusion



Data Availability






Acknowledgments



References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















bait tests


Immersion LC50= 2037 1697-2457 4158 3544

LC90= 4684 3686-6712

Bait LC50= 310 129-516 4760 2319

LC90= 1108 704-1700


50and 965 ppm LC













50and LC











5 Conclusion



Data Availability






Acknowledgments



References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















5 Conclusion



Data Availability






Acknowledgments



References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Efficacy of Achyranthes aspera (L.) as a Molluscicidal Bait...

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Transcript of Efficacy of Achyranthes aspera (L.) as a Molluscicidal Bait...