Veterinary Parasitology 169 (2010) 399–403

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Short communication

The preparation of neem oil microemulsion (Azadirachta indica) and the

comparison of acaricidal time between neem oil microemulsion and
other formulations in vitro
Jiao Xu a,1, Qiao-Jia Fan a,1, Zhong-Qiong Yin a,c,d,1,*, Xu-Ting Li b,1, Yong-Hua Du a,1,
Ren-Yong Jia a,c, Kai-Yu Wang a, Cheng Lv a, Gang Ye a, Yi Geng a, Gang Su a, Ling Zhao a,
Ting-Xiu Hu a, Fei Shi a, Li Zhang a, Chang-Long Wu a, Cui Tao a, Ya-Xue Zhang a, Dong-Xia Shi a
a
College of Animal Medicine, Sichuan Agricultural University, Ya’an 625014, PR China
b
Sichuan Animal Science Academy, Chengdu 610066, PR China
c
Key laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, PR China
d
State Key Laboratory of Veterinary Etiologocal Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, PR China

A R T I C L E I N F O A B S T R A C T

Article history: The preparation of neem oil microemulsion and its acaricidal activity in vitro was
Received 18 July 2009 developed in this study. In these systems, the mixture of Tween-80 and the sodium
Received in revised form 25 December 2009 dodecyl benzene sulfonate (SDBS) (4:1, by weight) was used as compound surfactant; the
Accepted 15 January 2010 mixture of compound surfactant and hexyl alcohol (4:1, by weight) was used as emulsifier
system; the mixture of neem oil, emulsifier system and water (1:3.5:5.5, by weight) was
Keywords: used as neem oil microemulsion. All the mixtures were stired in 800 rpm for 15 min at
Neem oil 40 8C. The acaricidal activity was measured by the speed of kill. The whole lethal time
Microemulsion
value of 10% neem oil microemulsion was 192.50 min against Sarcoptes scabiei var. cuniculi
Acaricidal activity
larvae in vitro. The median lethal time value was 81.7463 min with the toxicity regression
In vitro
equations of Y = 6.0269 + 3.1514X. These results demonstrated that neem oil micro-
emulsion was effective against Sarcoptes scabie var. cuniculi larvae in vitro.
Crown Copyright ß 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction surfactant film; the penetration and association of the

Microemulsions (ME) are clear, thermodynamically
stable, isotropic mixture of oil, water, and surfactant,
frequently in combination with a cosurfactant (Lawrence
and Rees, 2000). The structure of microemulsion consists
of a disordered, connected surfactant monolayer separat-
ing the two solvent domains. The conditions of micro-
emulsion formation are as follows: very low interfacial
tension at the water–oil interface; a highly fluid interfacial
* Corresponding author.
E-mail address: yinzhongq@163.com (Z.-Q. Yin).
1
These authors contribute equally to this work and should be
considered as the first author.
molecules of the oil phase with the interfacial surfactant
film (Kreilgaard, 2002). Up to date microemulsions have
been shown to be able to protect labile drug, control drug
release, increase drug solubility, and reduce patient
variability (Gülten et al., 2007). Due to its advantages,
microemulsion has become a potential system for both
hydrophilic and hydrophobic drugs (Kogan and Garti,
2006).
Neem oil extracted from the seeds of Azadirachta indica
has versatile medicinal properties, including anti-fertility,
antifungal, antibacterial, immunostimulant, antipyretic
(Biswas et al., 2002) and acaricidal activities (Mulla and
Su, 1999). As an acaricide, neem oil is effective against ticks
(Al-Rajhy et al., 2003; Garboui et al., 2006), poultry red
mites (Lundh et al., 2005) and S. scabiei (Du et al., 2007). In

0304-4017/$ – see front matter . Crown Copyright ß 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2010.01.016
400 J. Xu et al. / Veterinary Parasitology 169 (2010) 399–403
Fig. 1. The structure of octadecanoic acid-tetrahydrofuran-3,4-diyl ester.
a previous study we found that chloroform extracts of
neem oil exhibited potent acaricidal activity against S.
scabiei var. cuniculi larvae. A new component, octadecanoic
acid-tetrahydrofuran-3,4-diyl ester (Fig. 1), a kind of white
powder, had been isolated from neem oil, with a median
lethal concentration (LC50) of 0.1 mg/mL at 24 h post-
treatment. The median lethal time (LT50) for this com-
pound was 15.3 h at a concentration of 7.5 mg/mL (Du et
al., 2008, 2009).
The aim of this study is to develop and compare
appropriate microemulsion formulations as drug carriers.
In this case, the preparation of neem oil microemulsion and
its acaricidal activity in vitro were discussed.
2. Material and methods
2.1. Materials
Neem oil which was extracted from the seeds of the
neem by using CO2 supercritical fluid extraction was
supplied by a pesticide company (Green Gold Biological
Science & Technology Co. Ltd., Chengdu, PR China). All
chemicals we used in the test were analytical reagent
(AR  99.7%).
S. scabiei mites were isolated from infested rabbits. The
scabs, which were collected from the infested legs, were
placed in petri dishes and transported to a laboratory
within 2 h. Subsequently, all the petri dishes were
incubated at 35 8C for 30 min. Under the stereomicroscope,
according to Walton and Currie (2007), the motile larvae of
the S. scabiei mites are easy distinguished with the nymph
were used in the experiments.
2.2. Preparation of emulsifier system
Tween-80 and Sodium dodecyl benzene sulfonate
(SDBS) were used as compound surfactants. Tween-80
and SDBS were mixed at the weight ratio of 4:1, then
adding the different cosurfactants (amyl alcohol, hexyl
alcohol or heptyl alcohol) to the mixture respectively to
make emulsifier systems; the weight ratio of the com-
pound surfactants to cosurfactant was 4:1.
2.3. Construction of pseudo-ternary phase diagrams
In order to determine the best emulsifier system for the
preparation of the neem oil microemulsion, pseudo-
ternary phase diagrams were constructed using water
titration method at 40 8C (Pons et al., 2003). For each phase
diagram, the ratios of the emulsifier system to neem oil
were varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9.
Table 1
Factors and levels of orthogonal design.
Factor A B (8C) C (rpm) D (min)
Levels 1 2.5 25 800 10
2 3.0 40 1600 15
3 3.5 55 2400 20
Note: A, the weight ratio of emulsifier system to neem oil; B, the
temperature of preparation; C, the rate of stirring; D, the time of stirring.
Water was added drop-wisely into the mixtures. The phase
boundary was determined by observing the changes of the
sample appearance being from transparent to turbid.
2.4. Optimization of preparation process
The preparation neem oil microemulsion was optimized
by orthogonal test. 4 factors was described in Table 1: the
weight ratio of emulsifier system to neem oil, the
temperature of preparation, the rate and the time of stirring.
Experiment was designed according to the L9(34) orthogonal
table. In the experiment, every factor was three levels.
Viscosity and transparency were used as indicators (Guan et
al., 2004). Transparency was judged according to the
following standards: transparency, translucence, and opa-
city scored 10, 5, and 0 respectively; viscosity was judged as
follows: low viscosity (the same as water), medium
viscosity, and high viscosity scored 10, 5, and 0 respectively.
2.5. Characterization and stability of microemulsions
Sudan Red and Methylene Blue were used to judge the
type of the microemulsions. If the red diffuse faster than
the blue, the microemulsion was W/O. On the contrary, the
blue diffuse faster than red, it was O/W. The micromulsion
was observed by transmission electron microscope (TEM).
The viscosity was determined by rotation viscosimeter. All
of above measurements were carried out at 25 8C.
The centrifuge tests were carried out to assess the
physical stability of microemulsion. It was centrifuged for
5 h at 4000 rpm and for 20 min at 10,000 rpm in the
centrifuge test. Microemulsion was sealed in tube and
stored at 4  1 8C, 20  1 8C, 54  1 8C for 15 d and stored in
room temperature for 6 months. Then the characterizations
of the micromulsion were measured to judge the stability.
2.6. Preparation of neem oil aqueous emulsion and liquid
paraffin Solution
The neem oil aqueous emulsion was prepared as
follows: neem oil 10 g, Tween-80 3 g, distilled water
5 mL were grinded rapidly. Then the mixture was diluted
to 100 mL with distilled water.
10 g of neem oil was diluted to 100 mL with liquid
paraffin to form the liquid paraffin neem oil.
2.7. Assay of acaricidal activity in vitro
Acaricidal activity was assayed as previously reported
(Fichi et al., 2007) with slight modification. Larval mites
(n = 20) were placed in small polystyrene plates (8.5 mm in
 J. Xu et al. / Veterinary Parasitology 169 (2010) 399–403 401

diameter and 0.3 mm deep) and 2 mL of 10% neem oil
microemulsion, 10% neem oil aqueous emulsion and 10%
liquid paraffin neem oil were added to separate plates.
Distilled water and the all of the preparations without
neem oil were used as controls. The study was performed
in triplicate. All plates were placed in a humidity chamber
(75% relative humidity) at 25 8C and observed under a
stereomicroscope every 10 min until all the mites died,
and then calculated the values of the whole lethal time
and the median lethal time. Persistent immobility of larval
mites, even when stimulated with a needle, lack of
reaction, was considered indicative of death (Macchioni
et al., 2004).
2.8. Statistical analyses
The data was evaluated using the statistical analysis
software SPSS12.0. The significance differences of the
whole lethal time value were analyzed and the LT50
value was calculated by Probability method (An et al.,
2002).
3. Results
3.1. Pseudo-ternary phase diagrams
The pseudo-ternary phase diagrams with various
cosurfactants were shown in Fig. 2. The transparent
microemulsion region was presented in phase diagrams.
It can be seen that the region of microemulsion including
hexyl alcohol is bigger to that of amyl alcohol and heptyl
alcohol. Therefore, hexyl alcohol was the best one in the
three cosurfactants to form neem oil microemulsion.
3.2. Preparation of neem oil microemulsions
The results of the orthogonal test were shown in
Table 2.Through the orthogonal test, neem oil, emulsifier

Fig. 2. Pseudo-ternary phase diagrams of different neem oil microemulsions: (a) Tween-80/SDBS/amyl alcohol/neem oil/water, (b) Tween-80/SDBS/hexyl
alcohol/neem oil/water, (c) Tween-80/SDBS/heptyl alcohol/neem oil/water. The region of microemulsion including hexyl alcohol is bigger to that of amyl
alcohol and heptyl alcohol.
402 J. Xu et al. / Veterinary Parasitology 169 (2010) 399–403

Table 2 were globular and diffused uniformly. The viscosity of the

The result of orthogonal experiment.
microemulsion was 9.960 mPa s at 25 8C.
Test no. A B C D Score The centrifuge tests showed that the neem microemul-
1 1 1 1 1 5 sion had good physical stability. The phase separation was
2 1 2 2 2 5 not observed when the microemulsion stored at 4  1 and
3 1 3 3 3 0 54  1 8C for 15 d.It froze at 20  1 8C. But it recovered clear in
4 2 1 2 3 15 room temperature for a few minutes. After being stored in
5 2 2 3 1 15
room temperature for 6 months, the microemulsion was still
6 2 3 1 2 0
7 3 1 3 2 15 clear.
8 3 2 1 3 20
9 3 3 2 1 20 3.4. The whole lethal time
K1 10 35 25 40
K2 30 40 40 20
K3 55 20 30 35 The results were shown in Table 3. The whole lethal
k1 3.33 11.67 8.33 13.33 time value of 10% neem oil microemulsion, 10% neem oil
k2 10 13.33 13.33 6.67 aqueous emulsion and 10% paraffinic neem oil was 192.50,
k3 18.33 6.67 10 11.67 212.50, and 337.50 min respectively.
R 15 6.66 5 6.66

Note: A, the weight ratio of emulsifier system to neem oil; B, the 3.5. The median lethal time
temperature of preparation; C, the rate of stirring; D, the time of stirring.

The results were shown in Table 4. The median lethal

time (LT50) of 10% neem oil microemulsion,10% neem oil
Table 3 aqueous emulsion and 10% paraffinic neem oil was 81.75,
The whole lethal time of Sarcoptes scabie var treated with different neem
95.55, 156.68 min, with the toxicity regression equations of
oil formulations.
Y = 6.0269 + 3.1514X, Y = 9.9784 + 5.0390X, Y = 11.4068 +
Formulation The The whole lethal 5.1967X respectively. These results demonstrated that,
number time (min)
compared with the 10% paraffinic neem oil, neem oil
of mite
microemulsion had stronger effect against Sarcoptes scabiei
10% neem oil microemulsion 20 192.50  12.93ebc var. At the same time, the microemulsion without neem oil
Microemulsion without neem oil 20 231.25  9.87bc
and the 10% neem oil aqueous emulsion also showed some
10% neem oil aqueous emulsion 20 212.50  22.87bc
Aqueous emulsion without neem oil 20 437.50  31.91bc acaricidal activity.
10% paraffinic neem oil 20 337.50  37.06ac
Paraffinic solution 20 2495.00  268.37c 4. Discussion
Distilled water 20 1200.00  0.00b

Note: aSignificant difference between each neem oil formulation and its
blank formulation; bsignificant difference between each group and the
blank paraffinic solution; csignificant difference between each group and
distilled water; dsignificant difference between each neem oil formula-
tion and neem oil aqueous emulsion; esignificant difference between each
neem oil formulation and paraffinic neem oil solution.
system and water were mixed at the weight ratio of
1:3.5:5.5. Every preparative step of the formulation was
performed by stirring with a magnetic stirrer at 800 rpm
for 15 min at 40 8C.
3.3. Characterization and stability of microemulsion
The results showed that the type of the neem oil
microemulsion was O/W. Through the TEM, we can
observe that all droplets of the neem oil micromulsion
Oil emulsion is wildly used in pesticide. It contains large
number of organic solvents which are harmful to
environment. Because of their adverse effects on ecosys-
tem, many studies have concentrated on the O/W
emulsion. The neem oil microemulsion we had prepared
in this test was identified as the O/W microemulsion.
Water was used as medium, so the amount of organic
solvents was dramatically decreased.
Neem oil could be used for controlling parasitic mites
on the sheep body surface (Dimri and Sharma, 2003). It had
been reported by Hirudkar et al. (1997) that: 87.8% S.
scabiei mites that infested in sheep have been controlled by
50% neem oil when post-treatment 30 d. Sinha et al. (2004)
demonstrated that in pigs infested with S. scabiei mites
there was 100% cure by day 13 post-treatment with a
herbal-mineral preparation (50 mL neem oil, 50 mL karanj

Table 4
The median lethal time of Sarcoptes scabie var treated with different neem oil formulations.

Formulation Toxicity regression LT50 (min) The 95% confidence limit x2 Correlation
equations coefficient

10% neem oil microemulsion Y= 6.0269 + 3.1514X 81.7463 70.409–92.5580 5.3100 0.960
Microemulsion with out neem oil Y= 10.0943 + 5.1773X 89.0666 80.4341–97.2366 3.1210 0.970
10% neem oil aqueous emulsion Y= 9.978 4 + 5.0390X 95.5515 86.4395–104.2472 4.5350 0.941
Aqueous emulsion without neem oil Y= 7.9054 + 3.5633X 165.4010 150.3219–181.8938 11.7790 0.986
10% liquid paraffin Neem oil solution Y= 11.4068 + 5.1967X 156.6799 144.8752–169.8266 8.5390 0.976
liquid paraffin Y= 6.1434 + 1.9212X 1576.1946 609.4754–47790664.1621 2.9570 0.904
 J. Xu et al. / Veterinary Parasitology 169 (2010) 399–403
oil, and 10 g camphor). It was also having a good effect on
the control of the Dermanyssus gallinae (92% lethality rate)
by four weeks post-treatment with 20% of the neem oil
emulsion in the clinical trials (Lundh et al., 2005). The
previous studies in our laboratory showed that neem oil
had obvious effect against S. scabiei var. cuniculi larvae in
vitro. The value of LC50 and LC95 were 2.908 mL/mL and
12.018 mL/L by 24 h post-treatment (Du et al., 2007). The
current study demonstrated that 10% neem oil micro-
emulsion had high-performance acaricidal activity against
S. scabie var. cuniculi larvae in vitro.
As a control sample, the microemulsion without neem
oil in this test also showed some activity to kill mites,
which may be due to SDBS in the mixture. The studies of
Wu et al. (2005) showed that SDBS played a significant role
in killing human demodex in vitro. All the tested mites died
in 5 h with the SDBS concentration of 2%. Clinical trials
showed that the effective rate of 2% SDBS cream in
remedying human demodex was 87.1% (Zang et al., 2005).
As pesticide synergist, these surfactants which possess
activities on desinsection have been widely used to
enhance the effects of the drug, reduce the usage and so on.
In the test, we found that the 10% neem oil aqueous
emulsion also showed some acaricidal activity. The differ-
ence between the aqueous emulsion and the microemulsion,
their further activities against S. scabiei var. cuniculi and the
environmental toxicity will be the object of intensive study.
And increasing the dosage of the neem oil in the formulation
is also an important research content in the future.
Acknowledgements
The research was supported by Program for Changjiang
Scholars and Innovative Research Team in University
(PCSIRT0848), Sichuan Agricultural University Youths
Fund (2006C18), and the State Key Laboratory of Veter-
inary Etiologocal Biology, Lanzhou Veterinary Research
Institute, Chinese Academy of Agricultural Sciences
(SKLVEB2009KFKT022).
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