research article larvicidal activity of nerium oleander against

Research ArticleLarvicidal Activity of Nerium oleander against Larvae West NileVector Mosquito Culex pipiens (Diptera Culicidae)

Fouad El-Akhal12 Raja Guemmouh2

Yassine Ez Zoubi3 and Abdelhakim El Ouali Lalami14

1Regional Diagnostic Laboratory of Epidemiological and Environmental Hearth Regional Health DirectorateEL Ghassani Hospital 30000 Fez Morocco2Sidi Mohamed Ben Abdellah University Faculty of Sciences Dhar El Mahraz Laboratory of Biotechnology andPreservation of Natural Resources 30000 Fez Morocco3Laboratory of Phytochemistry National Institute of Medicinal and Aromatic Plants 34000 Taounate Morocco4Institute of Nursing Professions and Health Techniques EL Ghassani Hospital 30000 Fez Morocco

Correspondence should be addressed to Abdelhakim El Ouali Lalami eloualilalamiyahoofr

Received 28 July 2015 Revised 9 October 2015 Accepted 21 October 2015

Academic Editor Emmanuel Serrano Ferron

Copyright copy 2015 Fouad El-Akhal et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Outbreaks of the West Nile virus infection were reported in Morocco in 1996 2003 and 2010 Culex pipiens wasstrongly suspected as the vector responsible for transmission In the North center of Morocco this species has developed resistanceto synthetic insecticides There is an urgent need to find alternatives to the insecticides as natural biocides Objective In this workthe insecticidal activity of the extract of the local plant Nerium oleander which has never been tested before in the North center ofMorocco was studied on larval stages 3 and 4 of Culex pipiensMethods Biological tests were realized according to a methodologyinspired from standardWorld Health Organization protocolThemortality values were determined after 24 h of exposure and LC

50

and LC90values were calculated Results The extract had toxic effects on the larvae of culicid mosquitoes The ethanolic extract

of Nerium oleander applied against the larvae of Culex pipiens has given the lethal concentrations LC50and LC

90in the order of

5757mgmL and 16635mgmL respectively Conclusion This investigation indicates that N oleander could serve as a potentiallarvicidal effective natural biocide against mosquito larvae particularly Culex pipiens

1 Introduction

The diseases vectored by mosquitoes continue to be a majorcause of illnesses and death worldwide [1 2] Malaria fila-riasis Japanese encephalitis and dengue fever are the mostapparent diseases (parasitic and viral) vectored by mosqui-toes of the genera Anopheles Culex and Aedes [3 4]

In the years 1996 2003 and 2010 and according toa research outbreaks of West Nile virus infection werereported in Morocco [5 6] Moreover the study stated thatthe Culex pipiens (C pipiens) was strongly suspected as thevector responsible for transmission [5ndash8]The intensity of thetransmission depends on factors linked to the parasite thevector the host human and the environment

Due to the lack of awareness among people early detec-tion and complete treatment of these diseases were very

difficult [9] One of the available methods for controlling themosquitoes is the use of synthetic insecticides these latteradversely affecting the environment by contaminating the airwater and soil [10]

In the North center of Morocco the species C pipiens hasdeveloped resistance to the synthetic insecticide Temephoswhich is usually used in an antilarval fight [11] Let us notethat this species C pipiens has developed equal resistance onother insecticides as Malathion Fenthion and Fenitrothion(unpublished data)Therefore there is an urgent need to findalternatives to the insecticides as natural herbal biocides

Different parts of plants contain a complex of chemicalswith a unique biological activity The hydroethanol extractof plants and their components are widely used in theprevention and treatment of some human diseases Variousextracts of plants have been also documented to exhibit

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2015 Article ID 943060 5 pageshttpdxdoiorg1011552015943060

2 Journal of Parasitology Research

acute toxic effects against insects including mosquitoesNerium oleander (N oleander) is flowering shrub of Dogbanefamily It is popularly used as an ornamental plant [12]Throughout history this plant has been used in medicineas an antibacterial anti-inflammatory and antinociceptivecompound [13]

This study has been carried out with the aim to assessthe larvicidal activity of hydroethanolic extract ofN oleanderon C pipiens The insecticidal activity of N oleander plantagainst C pipiens has never been studied before in the Northcenter of Morocco

2 Materials and Methods

21 Crop Plants and Ultrasound-Assisted Extraction A sam-ple collection (leaves stems and roots) of a local plant (Noleander) was conducted in April (2014) at the mountain ofTimezgana falling within the rural community of Timezgana(area of Taounate North center of Morocco) to an approxi-mate altitude of 800m

In a 500mL beaker 20 g of a dried plant powder wasmixed with 150mL of hexane The beaker was set in aSonicator brand ldquoELMArdquo a frequency of 35 kHz for 45minwith a temperature of 25∘C The extract was filtered throughWhatman paper and the recovered solvent was rejected Dry-ing the powder in a plant incubator at a temperature of 40∘Cfor 30mins the powder was reextracted again with ethanolat 80 for 45min under the same conditions The finalextract was recuperated from the mixture (ethanolwater)after filtration by Whatman paper and evaporation undervacuum at 40∘C on a rotary evaporator [14]

22 Phytochemical Screening The phytochemical constitu-ents existing in the hydroethanolics are tannins flavonoidssterols terpenes triterpenes coumarins leucoanthocyaninsand mucilages obtained using a simple qualitative analysismethod as described in the study [15 16]

23 Characteristics of Larval Site The collection of larvae ofC pipiens was performed in a breeding site located in theurban area of the city of Fez called Grand Canal (402maltitude 30∘0310158403710158401015840N and 5∘0810158403510158401015840 E) This site originatingfrom a hot spring is characterized by a very high densityof Culicidae larvae The warm water from a thermal springcalled Ain Lah promotes the proliferation of larvae of Cpipiens

24 Collecting Larvae of C pipiens Larvae were collectedusing a rectangular plastic tray that was inclined 45∘ withrespect to the water surface the resulting tension forceattracts the plate to the larvae The larvae gathered weremaintained in breeding in rectangular trays at an averagetemperature of 217∘C plusmn 2∘C in the Entomology Unit at theRegional Diagnostic Laboratory Epidemiological and Envi-ronmental Health (RDLEH) falling within Regional HealthDirectorate of Fez

25 Identification of Larvae The identification of morpho-logical characters of larvae has been determined using

the Moroccan key of identification of Culicidae [17] and theidentification software of mosquitoes of the MediterraneanAfrica [18]

26 Protocol of Larval Susceptibility Testing The susceptibil-ity tests were carried out in accordance with the standardprotocol developed by WHO in 2005 [19] From the initialextract (100mgmL stock solution) of plant concentrationsof 20 40 60 80 100 120 140 and 160mgmL were preparedPreliminary experiments were used to select a range of con-centrations for the tests previously mentioned 1mL of eachsolution prepared was placed in beakers containing 99mLof distilled water in contact with 20 larvae of stages 3 and 4the same number of larvae was placed in a beaker containing99mL of distilled water plus 1mL of ethanolThree replicateswere carried out for each dilution and for the control After24 hours of contact we counted the living and dead larvaeThe results of susceptibility testing were expressed in thepercentage of mortality versus the concentration of plantextract used If the percentage of mortality in control isgreater than 5 the percentage ofmortality in larvae exposedto the extract shall be corrected by using Abbottrsquos formula[20]

Mortality Corrected

= [

( Mortality Observed minus Mortality Control)(100 minus Mortality Control)

]

times 100

(1)

If the controlmortality exceeds 20 the test is invalid andmust be repeated

27 Data Processing For the data processing we used thelog-probit analysis (Windl version 20) software developedby CIRAD-CAMABIS [21] The analysis of the averages andstandard deviation was also performed by using the test ofanalysis of variance ANOVA

3 Results

As it is illustrated in Table 1 the phytochemical screen-ing of extract of N oleander grown in North center ofMorocco revealed a presence of flavonoids sterols terpenestriterpenes and coumarins components However tanninsleucoanthocyanins and mucilages were not detected

31 Variation in Mortality Rate The hydroethanolic extractofN oleander is usedThemortality rate ranged between 18and 100 (Figure 1) The lowest concentration necessary toachieve 100 mortality of larvae of C pipiens was evaluatedat 160mgmL

32 LC50 and LC90 Lethal Concentrations Figure 1 confirmsthe analysis performed to the order of effectiveness ofaqueous extracts tested The aqueous extract of N olean-der exhibits the lowest LC

50of 5757mgmL (equation of

Journal of Parasitology Research 3

Table 1 Phytochemical screening of hydroethanolic extract of plant N oleander

Plant Tannins Flavonoids Sterols andTerpenes Triterpenes Coumarins Leucaonthocyans Mucilages

Nerium oleander minus + + + + minus minus

(+) = present (minus) = absent chemical

18

3333

44445454

6666

7892909

100

040 60 80 100 120 140 160 Control20

Concentrations (mgmL)

0102030405060708090

100

Larv

al m

orta

lity

()

Figure 1 Percentage of mortality recorded in the test sensitivity byaqueous extract of a plant on C pipiens

the regression line 119884 = minus489649 + 278167 lowast 119883 calculatedChi2 23364) and LC

90= 16635mgmL (Table 2)

4 Discussion

It is a noteworthy fact that plants have been evolvingfor over 400 million years and have developed protectionmechanisms such as repellents and even insecticidal effectsto defend themselves against insect attack Many researchpapers have reported the efficiency of plant extracts againstmosquito larvae [22ndash24]

The results of phytochemical screening of the extract ofN oleander which we found are in accordance with otherstudies related to theNerium familyThe species of this familyproduce flavonoids coumarins and triterpenes [25 26]

The larvicidal activity observed among extracts of Noleander could be explained by the action or effect of phyto-chemical components flavonoids sterols terpenes triterpe-nes and coumarins

Flavonoids have a key role in stress response mechanismsin plants The adaptive role of flavonoids in plant self-protection against bacterial fungal and viral diseases as wellas insects starts to gain importance in the understandingof plant defense Flavonoids which act as antioxidants orenzyme inhibitors are involved in photosynthesis and cellu-lar energy transfer processes and may serve as precursors oftoxic substances [27 28] or have a pharmacological activity[27]

In Vellore City India the larvicidal activity ofN oleanderwas evaluated against Culex larvae 43 of mortality wasfound from the concentration of 3 (30mgmL) duringthe 24-hour exposure [12] In our work this percentage of

Table 2 Concentrations LC50

and LC90

lethal larvae of C pipiensafter 24 hours of exposure

Plant LC50

(mgmL) (Ll-Ul)lowast LC90

(mgmL) (Ll-Ul)lowast

N oleander 5757 (3675ndash7592) 16635 (11819ndash36579)lowastLl-Ul lower limit-upper limit

mortality was observed for a concentration of 60mgmL(which is the double of concentration found by the previousstudy)

In another work Madhuri et al in 2013 [29] found thatN oleander did not show any larvae mortality of Culex in 1aqueous extract whereas 100 ppm (01mgmL) ofN oleanderhad 74 mortality in 72 hours

The results obtained by El-Sayed and Ghada in 2014 [30]indicated that diethyl ether extract of N oleander leavesshould reduce the population dynamics of C pipiens withLC50

of 10500mgL (105mgmL) In this study we found5757mgmL for the LC

50 which is five times more than the

concentration obtained by El-Sayed and GhadaIn the North West of Morocco one preliminary evalua-

tion study of the larvicidal activity of the N oleander planton fourth-stage larvae (L4) of the species C pipiens wasconducted This study made by Aouinty et al in 2006 [31]who exhibited that the aqueous extract of this plant has beenineffective in terms of toxicity with a calculated LC

50of about

3130 plusmn 310mgL (313mgmL)Various factors as the environmental conditions the

extraction technique the drying the period and gatheringsites the agricultural practices the plant age [32ndash35] theconcentration of the extract the concentration of its activecomponents or even factors regarding the mosquito caninfluence the performance the physicochemical characteris-tics and the chemical composition of the extract

Thus Fakoorziba et al in 2015 [36] in southern Iranconcluded that there was a high to low lethal effect of extractsof N oleander leaves against mosquito larvae (DipteraCulicidae) Anopheles stephensi depending on the solventused chloroform petroleum benzene water and acetone

Comparing our results with the work mentioned previ-ously we can deduce that the LC

50obtained by the larvicidal

action of the N oleander plant grown in North East ofMorocco on the larvae of the C pipiens is relatively effectiveHowever if we compare our LC

50(5757mgmL=57570 ppm)

to that of a chemical larvicide for example Temephos whichwas found between 00065 and 00094 ppm against C pipiens[11] we can deduce that our LC

50is far too high to be

effective against mosquito larvae Consequently the plant


extract should not really be considered an effective larvicideagainst mosquito

That is why the extracts of the aqueous phase of theN oleander grown in North center of Morocco should besubjected to separation to isolate and concentrate the activesubstances which certainly can presentmuch lower LC

50and

would be valued as an alternative insecticideOn the other hand several studies have reported that

all parts of the oleander plant including the sap dried orboiled are poisonous to humans animals fish birds and inparticular certain insects [37ndash40] Indeed the leaves contain amixture of a very toxic cardiac glycosides of cardenolides likeoleandrin oleandrigenin digoxin digitonin digitoxigeninnerizoside neritaloside and odoroside [41 42]

5 Conclusion

This study concluded that the hydroethanolic extract of Noleander possesses larvicidal activity with values of LC

50and

LC90 of 5757mgmL and 16635mgmL respectively The

larvicidal activity of hydroethanolic extract of N oleanderon C pipiens could be due to the major components Thiswould include the flavonoids the sterols the terpenes thetriterpenes and the coumarins

This plant can be used as a biological remedy to controlthe mosquito population in the locality at risk particularlythe Culex mosquito which possesses great menace of deadlydisease transmission Further studies on isolation of activeconstituents from the extract and their biological activity onculicid mosquito are under investigation

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors thank everyone who has contributed to thiswork especially the proofreader Mr Ba Sidi El Idrissi anEnglish teacher

References

[1] S Din W Akram H A A Khan A Hussain and F HafeezldquoCitrus waste-derived essential oils alternative larvicides fordengue fever mosquito Aedes albopictus (Skuse) (CulicidaeDiptera)rdquo Pakistan Journal of Zoology vol 43 no 2 pp 367ndash372 2011

[2] R Pavela ldquoLarvicidal effects of some Euro-Asiatic plants againstCulex quinquefasciatus Say larvae (Diptera Culicidae)rdquo Para-sitology Research vol 105 no 3 pp 887ndash892 2009

[3] A A Rahuman A Bagavan C Kamaraj E Saravanan A AZahir and G Elango ldquoEfficacy of larvicidal botanical extractsagainst Culex quinquefasciatus Say (Diptera Culicidae)rdquo Para-sitology Research vol 104 no 6 pp 1365ndash1372 2009

[4] R Borah M C Kalita A Kar and A K Talukdar ldquoLarvicidalefficacy of Toddalia asiatica (Linn) Lam against two mosquitovectors Aedes aegypti and Culex quinquefasciatusrdquo AfricanJournal of Biotechnology vol 9 no 17 pp 2527ndash2530 2010

[5] J Figuerola R E Baouab R SoriguerO Fassi-Fihri F LlorenteandM A Jımenez-Clavero ldquoWest Nile virus antibodies in wildbirds Morocco 2008rdquo Emerging Infectious Diseases vol 15 no10 pp 1651ndash1653 2009

[6] H Fassil M El Harrak and J-L Marie ldquoEpidemiologicalaspects of west Nile virus infection in Moroccordquo Medecine etSante Tropicales vol 22 no 2 pp 123ndash125 2012

[7] T Changbunjong T Weluwanarak N Taowan P Suksai TChamsai and P Sedwisai ldquoSeasonal abundance and potentialof Japanese encephalitis virus infection in mosquitoes at thenesting colony of ardeid birds Thailandrdquo Asian Pacific Journalof Tropical Biomedicine vol 3 no 3 pp 207ndash210 2013

[8] P Calistri A Giovannini Z Hubalek et al ldquoEpidemiology ofWest Nile in Europe and in the Mediterranean basinrdquo OpenVirology Journal vol 4 pp 29ndash37 2010

[9] R Pavela ldquoLarvicidal effects of various Euro-Asiatic plantsagainst Culex quinquefasciatus Say larvae (Diptera Culicidae)rdquoParasitology Research vol 102 no 3 pp 555ndash559 2008

[10] M S Shivakumar R Srinivasan and D Natarajan ldquoLarvicidalpotential of some Indian medicinal plant extracts againstAedesaegypti (l)rdquo Asian Journal of Pharmaceutical and ClinicalResearch vol 6 no 3 pp 77ndash80 2013

[11] A El Ouali Lalami F El-Akhal N El Amri S Maniar and CFaraj ldquoState resistance of the mosquito Culex pipiens towardstemephos central Moroccordquo Bulletin de la Societe de PathologieExotique vol 107 no 3 pp 194ndash198 2014

[12] R Lokesh E Leonard Barnabas P Madhuri K Saurav andK Sundar ldquoLarvicidal activity of Trigonellafoenum and Neriumoleander leaves against mosquito larvae found in Vellore CityIndiardquoCurrent Research Journal of Biological Sciences vol 2 no3 pp 154ndash160 2010

[13] N Erdemoglu E Kupeli and E Yesilada ldquoAnti-inflammatoryand antinociceptive activity assessment of plants used as rem-edy in Turkish folk medicinerdquo Journal of Ethnopharmacologyvol 89 no 1 pp 123ndash129 2003

[14] Y Ezzoubi D Bousta M Lachkar and A Farah ldquoAntiox-idant and anti-inflammatory properties of ethanolic extractof Lavandula stoechas L from Taounate region in MoroccordquoInternational Journal of Phytopharmacology vol 5 no 1 pp 21ndash26 2014

[15] R Paris and A Nothis ldquoSur quelques plantes de nouvelleCaledonierdquoPlantesmedicinales Et Phytotherapie vol 4 pp 274ndash287 1996

[16] A Diallo Etude de la phytochimie et des activites biologique deSyzygium guineense Willd (Myrtaceae) [PhD thesis] 2005

[17] O Himmi M Dakki B Trari and M A El Agbani TheCulicidae of Morocco Identification Keys with Biological andEcological Data (Work of the Scientific Institute) vol 44 of Seriesdes Rapportd de lrsquoInstitute Scientifique Institut Scientifique deRabat Rabat Morocoo 1995

[18] J Brunhes A Rhaim Geoffroy and J P Hervy Mosquitoes oftheMediterraneanAfrica Software Identification and Education1 CD-ROM (Didactiques) IRD Paris France IPT TunisTunisia 2000

[19] WHO ldquoGuidelines for laboratory and field testing of mosquitolarvicidesrdquo Tech Rep Whocdswhopesgcdpp200513 2005

[20] W S Abbott ldquoA method of computing the effectiveness of aninsecticiderdquo Journal of Economic Entomology vol 18 no 2 pp265ndash267 1925

[21] M Giner M Vassal C Vassal F Chiroleuand and Z KouaikLogiciel CIRAD URBIMABIS Montpelier France 1999


[22] C H S D Phani DeepthiYadav N S P Bharadwaj M YEdukondalu C H Methushala and A Ravi Kumar ldquoPhyto-chemical evaluation of Nyctanthes arbor-tristis Nerium olean-der and Catharanthus roseusrdquo Indian Journal of Research inPharmacy and Biotechnology vol 1 no 3 2013

[23] R Santhi G Lakshmi A M Priyadharshini and L Anan-daraj ldquoPhytochemical screening of Nerium oleander leaves andMomordica charantia leavesrdquo International Research Journal ofPharmacy vol 2 no 1 pp 131ndash135 2011

[24] R Raveen K T Kamakshi M Deepa S Arivoli and S Ten-nyson ldquoLarvicidal activity ofNerium oleander L (Apocynaceae)flower extracts against Culex quinquefasciatus Say (DipteraCulicidae)rdquo International Journal of Mosquito Research vol 1pp 38ndash42 2014

[25] M Roni K Murugan C Panneerselvam J Subramaniam andJ-SHwang ldquoEvaluation of leaf aqueous extract and synthesizedsilver nanoparticles using Nerium oleander against Anophelesstephensi (Diptera Culicidae)rdquo Parasitology Research vol 112no 3 pp 981ndash990 2013

[26] M M Sedaghat A Sanei Dehkordi M R Abai et al ldquoLarvi-cidal activity of essential oils of apiaceae plants against malariavectorAnopheles stephensirdquo Iranian Journal of Arthropod-BorneDiseases vol 5 no 2 pp 51ndash59 2011

[27] J B Harborne and T J Mabry The Flavonoids Advances inResearch Chapman amp Hall New York NY USA 1982

[28] J W McClure ldquoPhysiology of flavonoids in plantsrdquo in PlantFlavonoids in Biology and Medicine Biochemical Pharmacolog-ical and Structure Activity Relationships V Cody E Middletonand J B Harborne Eds pp 77ndash85 Alan R Liss New York NYUSA 1986

[29] P Madhuri R Lokesh and A R Revathy ldquolarvicidal activityof methanol extract of Nerium oleander and Trigonella foenumagainst Culex mosquito larvae along with a survey of entirevellore district for mosquitordquo International Journal of Pharmaand Bio Sciences vol 4 no 3 pp 574ndash580 2013

[30] H S El-Sayed and M E Ghada ldquoLarvicidal biological andgenotoxic effects and temperature-toxicity relationship of someleaf extracts of Nerium oleander (Apocynaceae) on Culex pipi-ens (Diptera Culicidae)rdquo Journal of Arthropod-Borne Diseasesvol 10 no 1 pp 1ndash11 2014

[31] B Aouinty S Oufara F Mellouki and S Mahari ldquoPreliminaryevaluation of larvicidal activity of aqueous extracts from leavesof Ricinus communis L and from wood of Tetraclinis articulata(Vahl) Mast on the larvae of four mosquito species Culexpipiens (Linne) Aedes caspius (Pallas) Culiseta longiareolata(Aitken) and Anopheles maculipennis (Meigen)rdquo BiotechnologyAgronomy and Society and Environment vol 10 no 2 pp 67ndash712006

[32] M Aberchane M Fechtal A Chaouch and T BouayouneldquoEffect of time and distillation techniques on essential oils yieldand quality of atlas cedar (Cedrus atlantica M)rdquo Annales de laRecherche Forestiere au Maroc vol 34 pp 110ndash118 2001

[33] M Bourkhiss M Hnach B Bourkhiss M Ouhssine AChaouch and B Satrani ldquoEffet de sechage sur la teneur etla composition chimique des huiles essentielles de Tetraclinisarticulata (Vahl) Mastersrdquo Agrosolutions vol 20 no 1 pp 29ndash44 2009

[34] O O Okoh A A Sadimenko and A J Afolayan ldquoThe effectsof age on the yield and composition of the essential oils ofCalendula officinalisrdquo Journal of Applied Sciences vol 7 no 23pp 3806ndash3810 2007

[35] M Bourkhiss M Hnach T Lakhlifi A Boughdad A Farahand B Satrani ldquoEffect of age and vegetative stage on essentialoil content and chemical composition of Thuya articulatardquo LesTechnologies de Laboratoire vol 6 no 23 pp 64ndash68 2011

[36] M R Fakoorziba M D Moemenbellah-Fard K Azizi andF Mokhtari ldquoMosquitocidal efficacy of medicinal plant Ner-ium oleander (Apocynaceae) leaf and flower extracts againstmalaria vector Anopheles stephensi Liston (Diptera Culicidae)larvaerdquoAsian Pacific Journal of Tropical Disease vol 5 no 1 pp33ndash37 2015

[37] V Gupta and P Mittal ldquoPhytochemical and pharmacologicalpotential of Nerium oleander a reviewrdquo International Journal ofPharmaceutical Sciences and Research vol 1 no 3 pp 21ndash272010

[38] SD Langford andP J Boor ldquoOleander toxicity an examinationof human and animal toxic exposuresrdquo Toxicology vol 109 no1 pp 1ndash13 1996

[39] F D Galey ldquoOleander (Nerium oleander) toxicosisrdquo in FoodAnimal Practice J L Howard Ed vol 4 of Current VeterinaryTheraphy pp 275ndash276 WB Saunders Philadelphia Pa USA1999

[40] G Tayoub H Sulaiman and M Alorfi ldquoAnalysis of oleandrinin oleander extract (Nerium oleander) by HPLCrdquo Journal ofNatural Products vol 7 pp 73ndash78 2014

[41] G E Trease and W C Evans Pharmacognosy WB SaundersEdinburg Tex USA 15th edition 2002

[42] S Tiwari and A Singh ldquoToxic and sub-lethal effects of olean-drin on biochemical parameters of fresh water air breathingmurrel Channa punctatus (Bloch)rdquo Indian Journal of Experi-mental Biology vol 42 no 4 pp 413ndash418 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


acute toxic effects against insects including mosquitoesNerium oleander (N oleander) is flowering shrub of Dogbanefamily It is popularly used as an ornamental plant [12]Throughout history this plant has been used in medicineas an antibacterial anti-inflammatory and antinociceptivecompound [13]

This study has been carried out with the aim to assessthe larvicidal activity of hydroethanolic extract ofN oleanderon C pipiens The insecticidal activity of N oleander plantagainst C pipiens has never been studied before in the Northcenter of Morocco

2 Materials and Methods

21 Crop Plants and Ultrasound-Assisted Extraction A sam-ple collection (leaves stems and roots) of a local plant (Noleander) was conducted in April (2014) at the mountain ofTimezgana falling within the rural community of Timezgana(area of Taounate North center of Morocco) to an approxi-mate altitude of 800m

In a 500mL beaker 20 g of a dried plant powder wasmixed with 150mL of hexane The beaker was set in aSonicator brand ldquoELMArdquo a frequency of 35 kHz for 45minwith a temperature of 25∘C The extract was filtered throughWhatman paper and the recovered solvent was rejected Dry-ing the powder in a plant incubator at a temperature of 40∘Cfor 30mins the powder was reextracted again with ethanolat 80 for 45min under the same conditions The finalextract was recuperated from the mixture (ethanolwater)after filtration by Whatman paper and evaporation undervacuum at 40∘C on a rotary evaporator [14]

22 Phytochemical Screening The phytochemical constitu-ents existing in the hydroethanolics are tannins flavonoidssterols terpenes triterpenes coumarins leucoanthocyaninsand mucilages obtained using a simple qualitative analysismethod as described in the study [15 16]

23 Characteristics of Larval Site The collection of larvae ofC pipiens was performed in a breeding site located in theurban area of the city of Fez called Grand Canal (402maltitude 30∘0310158403710158401015840N and 5∘0810158403510158401015840 E) This site originatingfrom a hot spring is characterized by a very high densityof Culicidae larvae The warm water from a thermal springcalled Ain Lah promotes the proliferation of larvae of Cpipiens

24 Collecting Larvae of C pipiens Larvae were collectedusing a rectangular plastic tray that was inclined 45∘ withrespect to the water surface the resulting tension forceattracts the plate to the larvae The larvae gathered weremaintained in breeding in rectangular trays at an averagetemperature of 217∘C plusmn 2∘C in the Entomology Unit at theRegional Diagnostic Laboratory Epidemiological and Envi-ronmental Health (RDLEH) falling within Regional HealthDirectorate of Fez

25 Identification of Larvae The identification of morpho-logical characters of larvae has been determined using

the Moroccan key of identification of Culicidae [17] and theidentification software of mosquitoes of the MediterraneanAfrica [18]

26 Protocol of Larval Susceptibility Testing The susceptibil-ity tests were carried out in accordance with the standardprotocol developed by WHO in 2005 [19] From the initialextract (100mgmL stock solution) of plant concentrationsof 20 40 60 80 100 120 140 and 160mgmL were preparedPreliminary experiments were used to select a range of con-centrations for the tests previously mentioned 1mL of eachsolution prepared was placed in beakers containing 99mLof distilled water in contact with 20 larvae of stages 3 and 4the same number of larvae was placed in a beaker containing99mL of distilled water plus 1mL of ethanolThree replicateswere carried out for each dilution and for the control After24 hours of contact we counted the living and dead larvaeThe results of susceptibility testing were expressed in thepercentage of mortality versus the concentration of plantextract used If the percentage of mortality in control isgreater than 5 the percentage ofmortality in larvae exposedto the extract shall be corrected by using Abbottrsquos formula[20]

Mortality Corrected

= [

( Mortality Observed minus Mortality Control)(100 minus Mortality Control)

]

times 100

(1)

If the controlmortality exceeds 20 the test is invalid andmust be repeated

27 Data Processing For the data processing we used thelog-probit analysis (Windl version 20) software developedby CIRAD-CAMABIS [21] The analysis of the averages andstandard deviation was also performed by using the test ofanalysis of variance ANOVA

3 Results

As it is illustrated in Table 1 the phytochemical screen-ing of extract of N oleander grown in North center ofMorocco revealed a presence of flavonoids sterols terpenestriterpenes and coumarins components However tanninsleucoanthocyanins and mucilages were not detected

31 Variation in Mortality Rate The hydroethanolic extractofN oleander is usedThemortality rate ranged between 18and 100 (Figure 1) The lowest concentration necessary toachieve 100 mortality of larvae of C pipiens was evaluatedat 160mgmL

32 LC50 and LC90 Lethal Concentrations Figure 1 confirmsthe analysis performed to the order of effectiveness ofaqueous extracts tested The aqueous extract of N olean-der exhibits the lowest LC

50of 5757mgmL (equation of






18

3333

44445454

6666

7892909

100

040 60 80 100 120 140 160 Control20


0102030405060708090

100

Larv

al m

orta

lity

()



90= 16635mgmL (Table 2)

4 Discussion







and LC90


Plant LC50











50of about







50(5757mgmL=57570 ppm)







50and



5 Conclusion


50and






Acknowledgments


References



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






18

3333

44445454

6666

7892909

100

040 60 80 100 120 140 160 Control20


0102030405060708090

100

Larv

al m

orta

lity

()



90= 16635mgmL (Table 2)

4 Discussion







and LC90


Plant LC50











50of about







50(5757mgmL=57570 ppm)







50and



5 Conclusion


50and






Acknowledgments


References



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




50and



5 Conclusion


50and






Acknowledgments


References



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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