Nanoemulsion a Potent Larvicidal Agent Againt Culex

8/10/2019 Nanoemulsion a Potent Larvicidal Agent Againt Culex

1/6

Research Article

Received: 1 November 2010 Revised: 22 April 2011 Accepted: 17 May 2011 Published online i n Wiley Online L ibrary:

(wileyonlinelibrary.com) DOI 10.1002/ps.2233

Neem oil (Azadirachta indica) nanoemulsion

a potent larvicidal agent against CulexquinquefasciatusCH Anjali, Yamini Sharma, Amitava Mukherjee andNatarajan Chandrasekaran

Abstract

BACKGROUND: Nanoemulsioncomposed of neem oiland non-ionicsurfactant Tween 20,with a meandroplet size ranging from31.03 to 251.43 nm, was formulated for various concentrations of the oil and surfactant. The larvicidal effect of the formulatedneem oil nanoemulsion was checked against Culex quinquefasciatus.

RESULTS: O/W emulsion was prepared using neem oil, Tween 20 and water. Nanoemulsion of 31.03 nm size was obtainedat a 1 : 3 ratio of oil and surfactant, and it was found to be stable. The larger droplet size (251.43 nm) shifted to a smallersize of 31.03 nm with increase in the concentration of Tween 20. The viscosity of the nanoemulsion increased with increasingconcentration of Tween 20. The lethal concentration (LC50) of the nanoemulsion against Cx. quinquefasciatus was checked for1 : 0.30, 1 : 1.5 and 1 : 3 ratios of oil and surfactant respectively. The LC50decreased with droplet size. The LC50for the ratio 1 : 3nanoemulsions was 11.75 mg L1.

CONCLUSION: The formulated nanoemulsion of 31.03 nm size was found to be an effective larvicidal agent. This is the first timethat a neem oil nanoemulsion of this droplet size has been reported. It may be a good choice as a potent and selective larvicidefor Cx. quinquefasciatus.c 2011 Society of Chemical Industry

Keywords: nanoemulsion; neem oil; ultrasonication;Culex quinquefasciatus

1 INTRODUCTIONNanoemulsions are submicron oil-in-water emulsions with ananoscale droplet diameter. They are thermodynamically stableand translucent dispersions of oil and water with a droplet sizein the range 100600 nm.1,2 Nanoemulsions are prepared bylow-energy methods such as phase inversion temperature (PIT)emulsification,3 by phase inversion composition4 or by highshear forces using high-pressure homogenisers or ultrasonicgenerators.5

Recently, nanoemulsions were identified as promising deliverysystems. Nanoemulsion preparation of natural oils could be apromising technology for mosquito repellents. Mosquitoes arevectors of malaria, filariasis and numerous viral diseases, suchas dengue, Japanese encephalitis and yellow fever.6 The useof repellents is important for preventing transmission of thesediseases to humans.

Neem(Azadirachta indica)isatraditionalplantofIndiawhichhasexcellentmedicinalandinsecticidalpropertiesduethepresenceofAzadirachtin.710 Okumuetal.11 prepared emulsions of neem oil,emulsifier and isopropanol. The larvicidal effect of the emulsionswas proved againstAnopheles gambiae.

There are not many reports on the nanoemulsion formulationof neem oil with a very low azadirachtin content. The presentwork focused on the nanoemulsion preparation of neem oil usinga high-energy method such as an ultrasonicator. This is one of

the extensively employed methods for nanoemulsion formationowing to its ease of use for large-scale production and low cost.5

The objective of the present study was to formulate andcharacterise nanoemulsions containing neem oil and non-ionicsurfactant Tween 20 with water as a continuous aqueous phase.The larvicidal potency of the formulated neem oil nanoemulsionwas checked against Culex quinquefasciatus. The authors wishedto establish whether emulsion droplet size influenced LC50.

2 EXPERIMENTAL METHODS

2.1 MaterialsNeemoilwasprocuredfromVellore,Tamilnadu,andstoredatroomtemperatureunderlabconditions.Tween20[polyoxyethylene(20)sorbitanmonolaurate]wassuppliedbySDFineChemLtd,Mumbai.Deionised water (Milli-Q water; Millipore Corporation) was usedfor all experiments. All chemicals were of analytical grade. The

Correspondence to: Natarajan Chandrasekaran, Centre for Nanobiotechnol-

ogy,School ofBiosciencesandTechnology,VIT University,Vellore632014,India.

E-mail: [email protected]

Centre for Nanobiotechnology, School of Biosciences and Technology, VIT

University,Vellore,India

Pest Manag Sci(2011) www.soci.org c 2011 Society of Chemical Industry


2/6

www.soci.org CH Anjalietal.

azadirachtin content in the procured neem oil was found to be0.068% w/v by HPLC.12

2.2 Larvae

Mosquito larvae were collected from a water stagnated area.Species identification was carried out in the Zonal EntomologicalResearch Laboratory, Vellore, Tamil Nadu, India. The mosquito

larvae were identified as Cx. quinquefasciatus. The larvae weremaintained under suitable temperature and humidity for acclima-tisation.

2.3 Construction of a phase diagram

Neemoil,Tween20andMilli-Qwaterwereusedinthepreparationof emulsion. Tween 20 was used as the surfactant becausenon-ionic surfactants are known to be less affected by pH andionic strength.13 The emulsions thus formed were sonicated for13 h using high-energy sonication in a sonicator (Ultrasonics,USA). A ternary phase diagram was constructed to optimise theconcentration of neem oil, Tween 20 and aqueous phase. Thenanoemulsions contained neem oil and surfactant in ratios of1 : 0.30, 1 : 0.50, 1 : 0.62, 1 : 0.66, 1 : 0.77, 1 : 1, 1 : 1.5, 1: 2, 1: 2.33,1 : 3, 1 : 4 and 1 : 5 respectively. Maximum ratios were covered forthe study to delineate the phase boundaries precisely formed inthe phase diagram at a temperature of 25 C. The physical stateof the nanoemulsion was marked on a ternary phase diagramwith one axis representing the oil, the second axis representingthe surfactant and the third axis representing the aqueous phase(Table 1).

2.4 Characterisation of nanoemulsions

2.4.1 Droplet size andsize distribution

Thedropletsizeandsizedistributionoftheneemoilnanoemulsionwere determined using a Brookhaven particle size analyser (90S).Nanoemulsions were diluted with water to reduce multiple

scattering effects prior to each experiment. Droplet size wasdescribed as the size in nm, and the polydispersity index (PDI)characterised the size distribution. Each measurementwas carriedout in triplicate.

2.4.2 Atomic force microscope (AFM)

TheshapeofneemoilnanoemulsionwasdeterminedusingAFM. 14

The nanoemulsion was diluted with water before it was used for

Table 1. Ten differentratiosof oil andsurfactant used fornanoemul-sion formulation

Oil : surfactant Size (nm)

1 : 0.30 251.43 0.58

1 : 0.50 238.9 0.12

1 : 0.62 223.3 0.15

1 : 0.66 208.0 0.058

1 : 0.77 207.7 0.12

1 : 1 192.1 0.058

1 : 1.5 93.0 0.33

1 : 2 74.5 0.088

1 : 2.33 46.3 0.088

1 : 3 31.03 1.73

1 : 4 31.03 0.79

1 : 5 31.02 0.68

AFM study. AFM studies were carried out by drop coating thediluted nanoemulsion onto a glass slide and drying in an oven. Itwas scanned at a rate of 100 mV s1 in the range 50 m 50 musing Nanosurf Easy Surf 2 (Switzerland).

2.4.3 Transmission electron microscopy (TEM)

The morphology of the nanoemulsion was determined by

transmission electron microscopy (TEM). For TEMstudies,a dropofnanoemulsion was placed on a copper grid and allowed to dry invacuum.15 Transmission electron micrographs were taken using aTecnaiG-10instrument(Philips), an80 kVTEM with a W-sourceandan ultrahigh-resolution pole piecewith a point-to-point resolutionof 1.9 .

2.4.4 Viscosity

The viscosity of the nanoemulsion was measured using a ThermoHaake RheoScope at 25 C. Experiments were performed intriplicate.

2.4.5 pHThe nanoemulsionpH was checked bya pHmeter (model HI 8417;Hanna Instruments Inc., Woonsocket, RI) at 20 1 C.

2.4.6 Stability of nanoemulsion

A stabilitystudy was performed by centrifuging the nanoemulsionat 3500 rpm for 30 min.16

The stability was also checked at refrigerator temperature (4 C)and room temperature (25 C).

2.4.7 Zeta potential measurements

The zeta potential of the nanoemulsion was determined using a

Brookhaven 90Plus zeta analyzer.

2.5 Larvicidal activity of the nanoemulsion

The larvicidal effect of the neem oil nanoemulsion was evaluatedfollowing the standard larval susceptibility test method.17 Na-noemulsionscontainingoilandsurfactantin a ratioof 1 : 0.30,1 : 1.5and 1 : 3 respectively wereselected forthe study. The droplet sizesof the nanoemulsion were considered as an important parameterfor the larvicidal study. Third-instar larvae ofCx. quinquefasciatuswere treated with different nanoemulsions. Controls were oneswithout treatments. Twenty larvae ofCx. quinquefasciatus wereplaced in a 250 mL sterile beaker containing 200 mL of water.After adding the larvae to the beaker, nanoemulsions of different

compositions were added to each of the beakers separately., Thebeakerscontainingthelarvaewerethenkeptatroomtemperaturein the growth room. The larvicidal effect of the nanoemulsion wasmonitored by recording mortality after 24 h of exposure. Each testwas performed with six replicates.

2.6 Statistical analysis

Valueswere expressedas themean standard deviation(SD). TheLC50 was determined at a 95% confidence level (P< 0.05) usingprobit analysis. The statistical significance of the difference wasexamined using two-way analysis of variance (ANOVA). Resultswith a probability value (P) of less than 0.05 were considered tobestatistically significant.

wileyonlinelibrary.com/journal/ps c 2011 Society of Chemical Industry Pest Manag Sci(2011)


3/6

Neem oil nanoemulsion as a larvicidal agent againstCx. quinquefasciatus www.soci.org

Figure 1. Ternary phase diagram of neem oil/Tween 20/water, showingthe O/W nanoemulsion area (shaded area).

3 RESULTS3.1 Ternary phase diagram

The ternary phase diagram of the three-component system ofwater,Tween20 andneemoil is shown inFig. 1. Tennanoemulsionformulations with different concentrations of neem oil and Tween20 were selected and were characterised. A continuous single-phase nanoemulsion region was observed over the oil with thewater weight content ranging from 0 to 100%. The area insidethe frame is the nanoemulsion region (Fig. 1). In this figure, onlynanoemulsion points are plotted (shaded area), so that there isno overcrowding of the phases in the diagram. The shaded areain the figure refers to the nanoemulsion region, while the outsidearea indicates multiphase turbid regions.

3.2 Characterization of nanoemulsions

3.2.1 Droplet size and size distribution

Droplet sizes of various formulated nanoemulsions are shown inTable 1. The droplet size decreasedwith increase in surfactantcon-centration (Fig. 2). The smallest droplet size of the nanoemulsionconsisting of onepart oil(6%) andthreepartssurfactant (18%) was31.03 1.73 nm. This nanoemulsion was stable ata 1 : 3 ratio. Thisis in accordance with data reported by Kale and Allen,18 accord-

ing to which the addition of surfactant to nanoemulsion systemscaused the interfacial film to condense and stabilize, resulting ina small droplet size. The polydispersity of the droplet size wasfound to be 0.211,0.364 and0.262 for nanoemulsionformulationswithoil and surfactantratiosof 1 : 0.30, 1 : 1.5 and 1 : 3 respectively.The size distribution of 1 : 3 nanoemulsions is shown in Fig. 3. Theeffective diameter was observed to be approximately 31 nm.

3.2.2 Atomic force microscope (AFM)

An AFM image of the nanoemulsion (1 : 3 ratio) stabilized byTween 20 is shown in Fig. 4. The shape of the nanoemulsion wasapproximately spherical in morphology. The mean diameter was48.2 nm.

Figure 2. Droplet size of nanoemulsions prepared using various ratios ofoil and surfactant.

Figure 3. Hydrodynamic size distribution of 1 : 3 nanoemulsions.

Figure 4. AFM image of neem oil nanoemulsion.

3.2.3 Transmissionelectron microscopy (TEM)

A TEM image of the nanoemulsion (1 : 3 ratio) is shown in Fig. 5.The diameter was 3570 nm (Fig. 5).

3.2.4 Viscosity

The viscosity of the selected formulations is shown in Table 2.The 1 : 0.30 nanoemulsion formulation showed lowest viscosity(1.670.003) comparedwith other formulations. This maybe dueto the increase in surfactant concentration.

Pest Manag Sci(2011) c 2011 Society of Chemical Industry wileyonlinelibrary.com/journal/ps


4/6


Figure 5. Transmission electron microscopic image of nanoemulsion.

3.2.5 pH

Withincreasein surfactantconcentration, thepH of thenanoemul-sionformulationincreased.Nanoemulsionformulationswithratiosof 1 : 0.30, 1: 1.5 and 1 : 3 showed pH values of 4.85, 5.55 and 5.60respectively (Table 2).

3.2.6 Stability of thenanoemulsion

The thermal stability of the nanoemulsion differentiates it fromemulsionswith kinetic stabilityand eventuallyphase separation.19

The steric effect plays an important role in stabilization of thenanoemulsion.20 The nanoformulation was found to be physicallystable at room temperature. Phase separation was not observed.The present results showed that the formulated nanoemulsionssurvived the stability tests.

3.2.7 Zeta potential measurements

The zeta potential of the nanoemulsion was between 26.87and 33.94 mV, confirming the stability of the formulatednanoemulsions.

Table 3. Mortality ofCulexquinquefasciatus mosquitolarvaeexposedto different droplet sizes of neem oil nanoemulsion

Mortality (%)

Concentration 1 : 0.30 1 : 1.5 1 : 3

1 ppm 0 0.21 0.85 0.21 3.37 0.17

5 ppm 6.77 0.21 18.49 0.17 38.66 0.17

10 ppm 17.80 0.17 33.62 0.17 53.79 0.17

25 ppm 27.97 0.17 47.90 0.21 57.98 0.21

50 ppm 38.14 0.17 58.83 0.17 68.91 0.17

100 ppm 48.31 0.17 73.11 0.21 86.56 0.21

Values are the mean of six values (n = mean SE).

Figure 6. A plot of size/LC50against various ratios of nanoemulsion.

3.3 Larvicidal activity of the nanoemulsion

Nanoemulsionformulationswithoil andsurfactantratiosof 1 : 0.30,1 : 1.5 and 1 : 3 were selected for larvicidal study. The percentage

mortalityfor third-instar larvaeofCx. quinquefasciatustreated withdifferent concentrations of neem oil nanoemulsion (ranging from1 to 100 mg L1) at the end of 24 h is shown in Table 3.

In the present investigation, the larvicidal activity of nanoemul-sions was studied againstCx. quinquefasciatus. The mortality rateofCx. quinquefasciatusincreases with increase in exposure time.The nanoemulsion formulation with a 1 : 3 ratio showed 86%mortality at 100 mg L1 concentration after 24 h of exposure.The nanoemulsion formulations with ratios of 1 : 1.5 and 1 : 0.30showed 73 and 48% mortality after 24 h of exposure. The larvi-cidal activity of neem oil alone was found to be relatively poor,whereas Tween 20 exhibited no larvicidal effect. The droplet sizesofnanoemulsionformulations withratiosof 1 : 3,1 : 1.5 and 1 : 0.30were 31.03, 93.0 and 251.43 nm respectively.

The LC50 of neem oil nanoemulsion of the smallest size(31.03 nm) was found to be 11.75 mg L1 for a 24 h exposureperiod (Fig. 6). The LC50 values of nanoemulsion formulations

Table 2. Droplet size, polydispersity and viscosity of nanoemulsion formulations

Ratio (oil : surfactant) Droplet size (nm)a Range Polydispersity Viscosity (mPa s)a pH

1 : 0.30 251.43 0.58 250.4251.43 0.211 0.004 1.67 0.003 4.85

1 : 1.5 93.0 0.33 92.593.0 0.364 0.002 2.0 0.006 5.55

1 : 3 31.03 1.73 29.931.03 0.262 0.10 3.68 0.003 5.60

a Mean SE,n = 3.



5/6

Neem oil nanoemulsion as a larvicidal agent againstCx. quinquefasciatus www.soci.org

with sizes of 93.0 nm and 251.43 nm were found to be 25.99 and62.89 mg L1 respectively. The LC50 decreased with droplet size.TheLC50 dataobtainedusingprobitanalysissoftwarerevealedthatthe tabulated results were significant, based on a 95% confidencelimit (P< 0.05). Students t-test and ANOVA were also found tobesignificant.

4 DISCUSSIONThe ternary phasediagram withtheoil/water(O/W)nanoemulsionarea is shown in Fig. 1. With increase in surfactant concentration,correspondingly the neem oil concentration decreased, whichresultedinasmalldropletsizeandreducedturbidity.Theemulsionturbidity is a function of particle concentration and size of theparticle.21

The stabilization of nanodroplets in the emulsion with a 1 : 3ratio of oil and surfactant would be due to the surfactant, whichreduces interfacial free energy and provides a mechanical barrierto coalescence.22 Polydispersity is a measure of the uniformityand stability of the droplet size in the formulation. Droplet sizein the nano range may be due to low values of polydispersity. 23

High polydispersity results in low uniformity of droplet size. Thesphericalform of thenanoemulsion andits size range (3070 nm),as revealed by AFM and TEM, are important findings. Similarresults were obtained by Mao etal.,14 who reported that thecharacterizationof-carotenenanoemulsion(stabilizedby Tween20) by AFM exhibited spherical morphology. In another set ofstudies by Baboota etal.,15 transmission electron microscopicstudies of celecoxibnanoemulsion exhibited a size range between19 and 78 nm. Hence, the present results are in substantialagreement with the above findings, and this might play animportant role in larvicidal study. It is possible that a reductionin droplet size, and hence an increase in surface area of thedroplets, increases the rate of accumulation by the larvae ofthe insecticidal component of the oil. This results in increasing

larvicidal efficacy of the nanoemulsion. Zebit24 reported that anallelochemicalsuchas azadirachtin actsas an anti-ecdysteroid andaffects the biochemical and physiological processes of the insectsystem, which results in larval death by the growth inhibitioneffect. They have also observed that azadirachtin nullifies theinsect detoxification mechanism, thereby reducing the selectionpressure for its development.

The negative charges on the droplets come from hydroxylion adsorption at the O/W interface, and the ethylene oxide (EO)groupsof Tween 80 maycreate hydrogen bonds with thehydroxylions to give more negative surface charges.25

Increase in the viscosity of the nanoemulsion with increase innon-ionicsurfactantparallelsthereportofEiniet al.,26 whichshows

that water molecules becometrapped in thecrosslinking chainsofthe non-ionic surfactant. This may be attributed to the increasedhydration by water molecules around the hydrophilic portion ofthe surfactants. Stachurski and Michalek27 have reported that thestability of emulsions can improve remarkably with increase in thesurface charge because of the repulsive forces produced betweendroplets against flocculation and coalescence.

Few reports are available on the larvicidal activity of crudeneem oil nanoemulsion with a low azadirachtin content againstCx. quinquefasciatus. Azadirachtin, nimbin, nimbidin and salanninare various triterpenoids present in neem oil. In the presentstudy, the azadirachtin concentration was only 0.068% w/v. Mostof the reported works have dealt with 0.15% w/v azadirachtin.This supports the finding by Virendra etal.28 that the larvicidal

activity of a neem oil formulation with 0.15% azadirachtin againstmosquitoes showed an LC50 of 1.8 mg L1. In a study by Batraetal.,29 various neem oil formulations were found to be effectivein controlling the proliferation ofAnopheles stephensiand Aedesaegyptiin pools and tanks for up to 23 weeks. This is the firsttime that a nanoemulsion formulation using neem oil with a smalldroplet size of 31.03 nm has been reported.

In the present study, neem oil nanoemulsion was found to be

effective in controlling mosquito larvae. The reduced size anduniform spreading of these fine particles increased the larvicidalefficacy. The nanoemulsion is easily affordable, economicallyfeasible and moreover less toxic than synthetic pesticides, andmaybe used as an alternative for control of vector-borne diseases.It has the advantage of being ecofriendly and effective, and hasshownpromisinglarvicidalactivity.However,furtherworkisgoingon in the authors laboratory to evaluate the topical applicationof neem-based nanoemulsions on human beings for mosquitorepellency.

5 CONCLUSIONS

The nanoemulsionformulation containing neemoil,Tween 20 anddeionized water was successfully optimized by the high-energymethod. A smallest droplet size of 31.03 nm was obtained. Neemoil nanoemulsion with the smallest droplet size was found to bemoreeffectiveincontrollingmosquitolarvaecomparedwithlargerdroplet sizes. Neem oil nanoemulsion may be a good alternativeto other pesticides for the control of vector-borne diseases.

ACKNOWLEDGEMENTSThe authors thank the management of VIT University forthe research fund extended by them for the completion ofthis work. They also thank Mr Rajagopal, senior entomologist,Zonal Entomological Research Laboratory, Vellore, Tamilnadu, for

identifyingCx. quinquefasciatusmosquito larvae.

REFERENCES1 Shafiq S,Faiyaz S,Sushma T, Farhan JA, Khar RKand Ali M, Designand

development of ramipril nanoemulsion formulation: in vitroandin vivoassessment.J Biomed Nanotechnol3:28 44 (2007).

2 Solans C, Esquena J,Forgiarini A,Uson N, Morales D,Izquierdo P, etal.,Absorption and aggregation of surfactants in solution, in Nano-emulsions: Formation, Properties and Applications, ed. by Mittal KLand Dinesh OS. Marcel Dekker, New York, NY, pp. 525 554 (2003).

3 Forster T, Von Rybinski W and Wadle A, Influences of microemulsionphases on the preparation of fine disperse emulsions. Adv ColloidInterface Sci58:119149 (1995).

4 Forgiarini A, Esquena J, Gonzalez C and Solans C, Formation of nano-

emulsions by low-energy emulsification methods at constanttemperature. Langmuir17:20762083 (2001).5 Solans C, Izquierdo P, Nolla J, Azemar N and Garcia-Celma MJ, Nano-

emulsions. Curr Opin Colloid Interface Sci10:102110 (2005).6 ElHag EA,Nadi AHand Zaitoon AA,Toxicand growthretardingeffects

of three plant extracts on Culex pipienslarvae (Diptera: Culicidae).Phytother Res13:388392 (1999).

7 Lucantoni L, Giusti F, Cristofaro M, Pasqualini L, Esposito F, Lupetti P,etal., Effects of neem extract on blood feeding, oviposition andoocyte ultra structure in Anopheles stephensi Liston (Diptera:Culicidae). Tissue Cell38:361 371 (2006).

8 Mordue AJand Nisbet AJ,Azadirachtinfrom theneem treeAzadirachtaindica:itsactionsagainstinsects. Ann EntomolSoc Brazil29:615632(2000).

9 Singh N, Mishra AK and Saxena A, Use of neem cream as a mosquitorepellent in tribal areas of central India. Indian J Malariol33:99102(1996).

Pest Manag Sci(2011) c 2011 Society of Chemical Industry wileyonlinelibrary.com/journal/ps


6/6


10 Su T and Mulla MS, Antifeedancy of neem products containingazadirachtin against Culex tarsalis and Culex quinquefasciatus(Diptera: Culicidae).J Vector Ecol23:114122 (1998).

11 Okumu FO, Knols BGJ and Fillinger U, Larvicidal effects of a neem(Azadirachta indica) oilformulationon themalaria vectorAnophelesgambiae.Malar J6:63 (2007).

12 Neem extract concentrate containing azadirachtin. Specification IS14299, Bureau of Indian Standards (BIS) (1995).

13 Zhong GG, Han GC and Hee JS, Physicochemical characterization

and evaluation of a microemulsions system for oral delivery ofcyclosporineA. IntJ Pharm161:75 86 (1998).14 Mao L, Xu D, Yang J, Yuan F, Gao Y and Zhao J, Effects of small and

large molecule emulsifiers on the characteristics of -carotenenanoemulsions prepared by high pressure homogenization. FoodTechnol Biotechnol47:336 342 (2009).

15 Baboota S, Shakeel F, Ahuja A, Ali J and Shafiq S, Design, developmentandevaluationofnovelnanoemulsionformulationsfortransdermalpotential of celecoxib.Acta Pharm57:315332 (2007).

16 Shafiq S and Shakeel F, Stability and self-nanoemulsification efficiencyof ramipril nanoemulsion containing labrasol and plurol oleique.Clin Res RegulatoryAffairs 27:7 12 (2010).

17 WHOguidelines forlaboratory andfieldtesting of mosquito larvicides,CDS/WHOPES/GCDPP/05.13 (2005).

18 Kale NJ and Allen AV, Studies on microemulsions using Brij-96 assurfactant and glycerin, ethylene glycol and propylene glycol ascosurfactants. IntJ Pharm 57:87 93 (1998).

19 Shinoda K andKunieda H,Phase propertiesof emulsions:PIT andHLB,inEncyclopedia of Emulsion Technology, Marcel Dekker, New York,NY (1983).

20 Tadros T, Izquierdo P, Esquena J and Solans C, Formation and stabilityof nano-emulsions.Adv Colloid Interfac108:303318 (2004).

21 Reddy SR and Fogler HS, Emulsion stability: determination fromturbidity.J Colloid Interface Sci79:101104 (1981).

22 Reiss H, Entropy-induced dispersion of bulk liquids.J Colloid InterfaceSci53:61 70 (1975).

23 Shinoda K andSaito H,The stabilityof O/Wtype emulsionsas functionsof temperature and the HLB of emulsifiers: the emulsification byPIT-method.J Colloid Interface Sci30:258263 (1969).

24 Zebit CPW, Effect of some crude and azadirachta-enriched neem(Azadirachta indica) seed kernel extracts of larvae ofAedes aegypti.Entomol ExpAppl35:11 16 (1984).

25 Liu W, Sun D, Li C, Liu Q and Xu J, Formation and stability of paraffinoil-in-water nano-emulsions prepared by the emulsion inversionpoint method.J Colloid Interface Sci303:557563 (2006).

26 Eini DIDE, Barry BWand Rhodes CT, Micellar size, shape and hydrationof long-chain polyoxyethylene nonionic surfactants. J ColloidInterface SciI54:348 351 (1976).

27 Stachurski J and Michalek M, The effect of the z potential on thestability of a non-polar oil-in-water emulsion. J Colloid Interface Sci184:433436 (1996).

28 Virendra KD, Akhiilesh CP, Kamaraju R, Ashish G, Trilochan S andAditya PD, Larvicidal activity of neem oil (Azadirachta indica)formulation against mosquitoes. Malar J8:124 (2009).

29 Batra CP, Mittal PK, Adak T and Sharma VP, Efficacy of neem wateremulsion against mosquito immatures. Indian J Malariol35:1521

(1998).


Documents

Nanoemulsion a Potent Larvicidal Agent Againt Culex