Research Article

Received: 1 November 2010

Revised: 22 April 2011

Accepted: 17 May 2011

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/ps.2233

Neem oil (Azadirachta indica) nanoemulsion

a potent larvicidal agent against Culex
quinquefasciatus
CH Anjali, Yamini Sharma, Amitava Mukherjee and
Natarajan Chandrasekaran
Abstract
BACKGROUND: Nanoemulsion composed of neem oil and non-ionic surfactant Tween 20, with a mean droplet size ranging from
31.03 to 251.43 nm, was formulated for various concentrations of the oil and surfactant. The larvicidal effect of the formulated
neem 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 obtained
at 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 smaller
size of 31.03 nm with increase in the concentration of Tween 20. The viscosity of the nanoemulsion increased with increasing
concentration of Tween 20. The lethal concentration (LC50 ) of the nanoemulsion against Cx. quinquefasciatus was checked for
1 : 0.30, 1 : 1.5 and 1 : 3 ratios of oil and surfactant respectively. The LC50 decreased with droplet size. The LC50 for the ratio 1 : 3
nanoemulsions 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 time
that a neem oil nanoemulsion of this droplet size has been reported. It may be a good choice as a potent and selective larvicide
for Cx. quinquefasciatus.
c 2011 Society of Chemical Industry


Keywords: nanoemulsion; neem oil; ultrasonication; Culex quinquefasciatus

INTRODUCTION

Nanoemulsions are submicron oil-in-water emulsions with a

nanoscale droplet diameter. They are thermodynamically stable
and translucent dispersions of oil and water with a droplet size
in the range 100600 nm.1,2 Nanoemulsions are prepared by
low-energy methods such as phase inversion temperature (PIT)
emulsification,3 by phase inversion composition4 or by high
shear forces using high-pressure homogenisers or ultrasonic
generators.5
Recently, nanoemulsions were identified as promising delivery
systems. Nanoemulsion preparation of natural oils could be a
promising technology for mosquito repellents. Mosquitoes are
vectors of malaria, filariasis and numerous viral diseases, such
as dengue, Japanese encephalitis and yellow fever.6 The use
of repellents is important for preventing transmission of these
diseases to humans.
Neem (Azadirachta indica) is a traditional plant of India which has
excellent medicinal and insecticidal properties due the presence of
Azadirachtin.7 10 Okumu et al.11 prepared emulsions of neem oil,
emulsifier and isopropanol. The larvicidal effect of the emulsions
was proved against Anopheles gambiae.
There are not many reports on the nanoemulsion formulation
of neem oil with a very low azadirachtin content. The present
work focused on the nanoemulsion preparation of neem oil using
a high-energy method such as an ultrasonicator. This is one of

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the extensively employed methods for nanoemulsion formation

owing to its ease of use for large-scale production and low cost.5
The objective of the present study was to formulate and
characterise nanoemulsions containing neem oil and non-ionic
surfactant Tween 20 with water as a continuous aqueous phase.
The larvicidal potency of the formulated neem oil nanoemulsion
was checked against Culex quinquefasciatus. The authors wished
to establish whether emulsion droplet size influenced LC50 .

EXPERIMENTAL METHODS

2.1 Materials
Neem oil was procured from Vellore, Tamilnadu, and stored at room
temperature under lab conditions. Tween 20 [polyoxyethylene (20)
sorbitan monolaurate] was supplied by SD Fine Chem Ltd, Mumbai.
Deionised water (Milli-Q water; Millipore Corporation) was used
for all experiments. All chemicals were of analytical grade. The

Correspondence to: Natarajan Chandrasekaran, Centre for Nanobiotechnology, School of Biosciences and Technology, VIT University, Vellore 632014, India.
E-mail: nchandrasekaran@vit.ac.in
Centre for Nanobiotechnology, School of Biosciences and Technology, VIT
University, Vellore, India

c 2011 Society of Chemical Industry

www.soci.org
azadirachtin content in the procured neem oil was found to be
0.068% 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 Entomological
Research Laboratory, Vellore, Tamil Nadu, India. The mosquito
larvae were identified as Cx. quinquefasciatus. The larvae were
maintained under suitable temperature and humidity for acclimatisation.
2.3 Construction of a phase diagram
Neem oil, Tween 20 and Milli-Q water were used in the preparation
of emulsion. Tween 20 was used as the surfactant because
non-ionic surfactants are known to be less affected by pH and
ionic strength.13 The emulsions thus formed were sonicated for
13 h using high-energy sonication in a sonicator (Ultrasonics,
USA). A ternary phase diagram was constructed to optimise the
concentration of neem oil, Tween 20 and aqueous phase. The
nanoemulsions contained neem oil and surfactant in ratios of
1 : 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 for
the study to delineate the phase boundaries precisely formed in
the phase diagram at a temperature of 25 C. The physical state
of the nanoemulsion was marked on a ternary phase diagram
with one axis representing the oil, the second axis representing
the surfactant and the third axis representing the aqueous phase
(Table 1).
2.4 Characterisation of nanoemulsions
2.4.1 Droplet size and size distribution
The droplet size and size distribution of the neem oil nanoemulsion
were determined using a Brookhaven particle size analyser (90S).
Nanoemulsions were diluted with water to reduce multiple
scattering effects prior to each experiment. Droplet size was
described as the size in nm, and the polydispersity index (PDI)
characterised the size distribution. Each measurement was carried
out in triplicate.
2.4.2 Atomic force microscope (AFM)
The shape of neem oil nanoemulsion was determined using AFM.14
The nanoemulsion was diluted with water before it was used for

Table 1. Ten different ratios of oil and surfactant used for nanoemulsion formulation
Oil : surfactant
1 : 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
1:5

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Size (nm)
251.43 0.58
238.9 0.12
223.3 0.15
208.0 0.058
207.7 0.12
192.1 0.058
93.0 0.33
74.5 0.088
46.3 0.088
31.03 1.73
31.03 0.79
31.02 0.68

CH Anjali et al.

AFM study. AFM studies were carried out by drop coating the
diluted nanoemulsion onto a glass slide and drying in an oven. It
was scanned at a rate of 100 mV s1 in the range 50 m 50 m
using 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 TEM studies, a drop of
nanoemulsion was placed on a copper grid and allowed to dry in
vacuum.15 Transmission electron micrographs were taken using a
Tecnai G-10 instrument (Philips), an 80 kV TEM with a W-source and
an ultrahigh-resolution pole piece with a point-to-point resolution
of 1.9 .
2.4.4 Viscosity
The viscosity of the nanoemulsion was measured using a Thermo
Haake RheoScope at 25 C. Experiments were performed in
triplicate.
2.4.5 pH
The nanoemulsion pH was checked by a pH meter (model HI 8417;
Hanna Instruments Inc., Woonsocket, RI) at 20 1 C.
2.4.6 Stability of nanoemulsion
A stability study was performed by centrifuging the nanoemulsion
at 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 evaluated
following the standard larval susceptibility test method.17 Nanoemulsions containing oil and surfactant in a ratio of 1 : 0.30, 1 : 1.5
and 1 : 3 respectively were selected for the study. The droplet sizes
of the nanoemulsion were considered as an important parameter
for the larvicidal study. Third-instar larvae of Cx. quinquefasciatus
were treated with different nanoemulsions. Controls were ones
without treatments. Twenty larvae of Cx. quinquefasciatus were
placed 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., The
beakers containing the larvae were then kept at room temperature
in the growth room. The larvicidal effect of the nanoemulsion was
monitored by recording mortality after 24 h of exposure. Each test
was performed with six replicates.
2.6 Statistical analysis
Values were expressed as the mean standard deviation (SD). The
LC50 was determined at a 95% confidence level (P < 0.05) using
probit analysis. The statistical significance of the difference was
examined using two-way analysis of variance (ANOVA). Results
with a probability value (P) of less than 0.05 were considered to be
statistically significant.

c 2011 Society of Chemical Industry

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Neem oil nanoemulsion as a larvicidal agent against Cx. quinquefasciatus

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Figure 2. Droplet size of nanoemulsions prepared using various ratios of

oil and surfactant.

Figure 1. Ternary phase diagram of neem oil/Tween 20/water, showing

the O/W nanoemulsion area (shaded area).

RESULTS

3.1 Ternary phase diagram

The ternary phase diagram of the three-component system of
water, Tween 20 and neem oil is shown in Fig. 1. Ten nanoemulsion
formulations with different concentrations of neem oil and Tween
20 were selected and were characterised. A continuous singlephase nanoemulsion region was observed over the oil with the
water weight content ranging from 0 to 100%. The area inside
the frame is the nanoemulsion region (Fig. 1). In this figure, only
nanoemulsion points are plotted (shaded area), so that there is
no overcrowding of the phases in the diagram. The shaded area
in the figure refers to the nanoemulsion region, while the outside
area 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 in
Table 1. The droplet size decreased with increase in surfactant concentration (Fig. 2). The smallest droplet size of the nanoemulsion
consisting of one part oil (6%) and three parts surfactant (18%) was
31.03 1.73 nm. This nanoemulsion was stable at a 1 : 3 ratio. This
is in accordance with data reported by Kale and Allen,18 according to which the addition of surfactant to nanoemulsion systems
caused the interfacial film to condense and stabilize, resulting in
a small droplet size. The polydispersity of the droplet size was
found to be 0.211, 0.364 and 0.262 for nanoemulsion formulations
with oil and surfactant ratios of 1 : 0.30, 1 : 1.5 and 1 : 3 respectively.
The size distribution of 1 : 3 nanoemulsions is shown in Fig. 3. The
effective 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 by
Tween 20 is shown in Fig. 4. The shape of the nanoemulsion was
approximately spherical in morphology. The mean diameter was
48.2 nm.
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Figure 3. Hydrodynamic size distribution of 1 : 3 nanoemulsions.

Figure 4. AFM image of neem oil nanoemulsion.

3.2.3 Transmission electron 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
(1.67 0.003) compared with other formulations. This may be due
to the increase in surfactant concentration.

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CH Anjali et al.

Table 3. Mortality of Culex quinquefasciatus mosquito larvae exposed

to different droplet sizes of neem oil nanoemulsion
Mortality (%)
Concentration
1 ppm
5 ppm
10 ppm
25 ppm
50 ppm
100 ppm

1 : 0.30

1 : 1.5

1:3

0 0.21
6.77 0.21
17.80 0.17
27.97 0.17
38.14 0.17
48.31 0.17

0.85 0.21
18.49 0.17
33.62 0.17
47.90 0.21
58.83 0.17
73.11 0.21

3.37 0.17
38.66 0.17
53.79 0.17
57.98 0.21
68.91 0.17
86.56 0.21

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

Figure 5. Transmission electron microscopic image of nanoemulsion.

Figure 6. A plot of size/LC50 against various ratios of nanoemulsion.

3.2.5 pH
With increase in surfactant concentration, the pH of the nanoemulsion formulation increased. Nanoemulsion formulations with ratios
of 1 : 0.30, 1 : 1.5 and 1 : 3 showed pH values of 4.85, 5.55 and 5.60
respectively (Table 2).

3.2.6 Stability of the nanoemulsion

The thermal stability of the nanoemulsion differentiates it from
emulsions with kinetic stability and eventually phase separation.19
The steric effect plays an important role in stabilization of the
nanoemulsion.20 The nanoformulation was found to be physically
stable at room temperature. Phase separation was not observed.
The present results showed that the formulated nanoemulsions
survived the stability tests.

3.2.7 Zeta potential measurements

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

3.3 Larvicidal activity of the nanoemulsion

Nanoemulsion formulations with oil and surfactant ratios of 1 : 0.30,
1 : 1.5 and 1 : 3 were selected for larvicidal study. The percentage
mortality for third-instar larvae of Cx. quinquefasciatus treated with
different concentrations of neem oil nanoemulsion (ranging from
1 to 100 mg L1 ) at the end of 24 h is shown in Table 3.
In the present investigation, the larvicidal activity of nanoemulsions was studied against Cx. quinquefasciatus. The mortality rate
of Cx. quinquefasciatus increases 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 : 0.30
showed 73 and 48% mortality after 24 h of exposure. The larvicidal activity of neem oil alone was found to be relatively poor,
whereas Tween 20 exhibited no larvicidal effect. The droplet sizes
of nanoemulsion formulations with ratios of 1 : 3, 1 : 1.5 and 1 : 0.30
were 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 exposure
period (Fig. 6). The LC50 values of nanoemulsion formulations

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

Ratio (oil : surfactant)
1 : 0.30
1 : 1.5
1:3
a

Droplet size (nm)a

Range

Polydispersity

Viscosity (mPa s)a

pH

251.43 0.58
93.0 0.33
31.03 1.73

250.4251.43
92.593.0
29.931.03

0.211 0.004
0.364 0.002
0.262 0.10

1.67 0.003
2.0 0.006
3.68 0.003

4.85
5.55
5.60

Mean SE, n = 3.

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Neem oil nanoemulsion as a larvicidal agent against Cx. quinquefasciatus

with sizes of 93.0 nm and 251.43 nm were found to be 25.99 and
62.89 mg L1 respectively. The LC50 decreased with droplet size.
The LC50 data obtained using probit analysis software revealed that
the tabulated results were significant, based on a 95% confidence
limit (P < 0.05). Students t-test and ANOVA were also found to be
significant.

DISCUSSION

The ternary phase diagram with the oil/water (O/W) nanoemulsion

area is shown in Fig. 1. With increase in surfactant concentration,
correspondingly the neem oil concentration decreased, which
resulted in a small droplet size and reduced turbidity. The emulsion
turbidity is a function of particle concentration and size of the
particle.21
The stabilization of nanodroplets in the emulsion with a 1 : 3
ratio of oil and surfactant would be due to the surfactant, which
reduces interfacial free energy and provides a mechanical barrier
to coalescence.22 Polydispersity is a measure of the uniformity
and stability of the droplet size in the formulation. Droplet size
in the nano range may be due to low values of polydispersity.23
High polydispersity results in low uniformity of droplet size. The
spherical form of the nanoemulsion and its size range (3070 nm),
as revealed by AFM and TEM, are important findings. Similar
results were obtained by Mao et al.,14 who reported that the
characterization of -carotene nanoemulsion (stabilized by Tween
20) by AFM exhibited spherical morphology. In another set of
studies by Baboota et al.,15 transmission electron microscopic
studies of celecoxib nanoemulsion exhibited a size range between
19 and 78 nm. Hence, the present results are in substantial
agreement with the above findings, and this might play an
important role in larvicidal study. It is possible that a reduction
in droplet size, and hence an increase in surface area of the
droplets, increases the rate of accumulation by the larvae of
the insecticidal component of the oil. This results in increasing
larvicidal efficacy of the nanoemulsion. Zebit24 reported that an
allelochemical such as azadirachtin acts as an anti-ecdysteroid and
affects the biochemical and physiological processes of the insect
system, which results in larval death by the growth inhibition
effect. They have also observed that azadirachtin nullifies the
insect detoxification mechanism, thereby reducing the selection
pressure for its development.
The negative charges on the droplets come from hydroxyl
ion adsorption at the O/W interface, and the ethylene oxide (EO)
groups of Tween 80 may create hydrogen bonds with the hydroxyl
ions to give more negative surface charges.25
Increase in the viscosity of the nanoemulsion with increase in
non-ionic surfactant parallels the report of Eini et al.,26 which shows
that water molecules become trapped in the crosslinking chains of
the non-ionic surfactant. This may be attributed to the increased
hydration by water molecules around the hydrophilic portion of
the surfactants. Stachurski and Michalek27 have reported that the
stability of emulsions can improve remarkably with increase in the
surface charge because of the repulsive forces produced between
droplets against flocculation and coalescence.
Few reports are available on the larvicidal activity of crude
neem oil nanoemulsion with a low azadirachtin content against
Cx. quinquefasciatus. Azadirachtin, nimbin, nimbidin and salannin
are various triterpenoids present in neem oil. In the present
study, the azadirachtin concentration was only 0.068% w/v. Most
of the reported works have dealt with 0.15% w/v azadirachtin.
This supports the finding by Virendra et al.28 that the larvicidal
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activity of a neem oil formulation with 0.15% azadirachtin against

mosquitoes showed an LC50 of 1.8 mg L1 . In a study by Batra
et al.,29 various neem oil formulations were found to be effective
in controlling the proliferation of Anopheles stephensi and Aedes
aegypti in pools and tanks for up to 23 weeks. This is the first
time that a nanoemulsion formulation using neem oil with a small
droplet 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 and
uniform spreading of these fine particles increased the larvicidal
efficacy. The nanoemulsion is easily affordable, economically
feasible and moreover less toxic than synthetic pesticides, and
may be used as an alternative for control of vector-borne diseases.
It has the advantage of being ecofriendly and effective, and has
shown promising larvicidal activity. However, further work is going
on in the authors laboratory to evaluate the topical application
of neem-based nanoemulsions on human beings for mosquito
repellency.

CONCLUSIONS

The nanoemulsion formulation containing neem oil, Tween 20 and

deionized water was successfully optimized by the high-energy
method. A smallest droplet size of 31.03 nm was obtained. Neem
oil nanoemulsion with the smallest droplet size was found to be
more effective in controlling mosquito larvae compared with larger
droplet sizes. Neem oil nanoemulsion may be a good alternative
to other pesticides for the control of vector-borne diseases.

ACKNOWLEDGEMENTS
The authors thank the management of VIT University for
the research fund extended by them for the completion of
this work. They also thank Mr Rajagopal, senior entomologist,
Zonal Entomological Research Laboratory, Vellore, Tamilnadu, for
identifying Cx. quinquefasciatus mosquito larvae.

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