World Journal of Pharmaceutical ReseaRch

Bhide et al. World Journal of Pharmaceutical Research

Volume 3, Issue 2, 2910-2917. Research Article ISSN 2277 – 7105

DEVELOPMENT OF MOSQUITO REPELLENT FORMULATIONS

AND EVALUATION FOR ITS ACTIVITY

* Ms.Shubhangi Sharad Bhide1, Ms.Babita Himmatrao More2, Ms. Suvarna Prabhakar

Gajare3, Mr. Sachin Vinayak Tembhurne4

1*, 2, 3
Lecturer in Pharmacy, Yadavrao Tasgaonkar Institute of Pharmacy, Karjat, MH, India.
4
Assistant Professor, Sudhakarrao Naik Institute of Pharmacy, Pusad, Amravati, MH, India.

ABSTRACT
Article Received on
10 January 2014, Essential oilhas been reported to have many pharmacological
Revised on 29 January 2014,
Accepted on 28 February activities, one of which is their property to repel the mosquitoes and
2014 insects.The marigold infused oil is reported to be antibacterial,
astringent, antifungal, used in insect bites etc.Mosquito repellent cream
*Correspondence for of different concentrations i.e. 5% and 7.5% , a combination of
Author
marigold oil with two essential oils i.e. clove oil and dill oil were
Ms.Shubhangi Sharad Bhide
formulated and evaluatedas described in the American Society for
Lecturer in Pharmacy,
Yadavrao Tasgaonkar Testing and Materials (ASTM) standard E951-83 Laboratory testing of
Institute of Pharmacy, non-commercial mosquito repellent formulation on the skin.All
Karjat, MH, India. formulations were subjected to sensory evaluation with respect to
color, fragrance, appearance, shine and ease of applications as well as
constant pH, clarity, homogeneityand spreadability.

Keywords: Mosquito repellents, Marigold infused oil, Skin sensitivity, Tagetes

sp.(Asteraceae), Anethum graveolens (Apiaceae), Eugenia Caryophyllata (Myrtaceae).

INTRODUCTION
With over many species of mosquitoes believed to be responsible for spreading diseases such
as yellow fever, dengue hemorrhagic fever, epidemic polyarthritis, encephalitis and
malaria.1,4According to the world Health Organization(WHO)such diseases causes more than
3 million deaths annually.2There are many treatments for Malaria and other mosquito
transmitted diseases but it is always better to prevent the disease. Hence, the term mosquito
and other repellent came in existence. Topical or other application of mosquito repellent
repels the mosquitoes to bit. Mosquito repellent can be prepares synthetically or naturally.

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However, most synthetic chemical repellents, especially DEET, can be readily absorbed
through the skin, causing many accidental poisonings, especially of children. They also can
poison wildlife.DDT in particular has been shown to be very harmful to the environment, and
DEET is suspected to be a carcinogen, teratogen and/or mutagen. Therefore, another,
preferably non-toxic, means of repelling insects is desired. Many natural Substances are
known to repel insects and mosquitoes.3

India represents a vast repository of diverse flora of considerable medicinal importance.

Many researchers have reported the bioactivity of (Essential oils)Eucalyptus sp,Cedrus
deodara, Cymbopogon sp.and Tagetes minuta against mosquitoes.4,5 Here the research work
is available to determine the bioactivity of essential oils directly on the adult mortality. The
present work evaluates bioactivity of the oils from three plants Tagetes sp.(Asteraceae),
Anethum graveolens (Apiaceae) and Eugenia Caryophyllata (Myrtaceae).Essential oil is very
important class of phytochemicals. We can see the use of essential oils in many herbal
products like turmeric in cosmetic and clove in dental. They have been reported to have many
pharmacological activities, one of which is their property to repel the mosquitoes and insects.
It has been proved that essential oils like cinnamon oil, peppermint oil, etc are good mosquito
repellents.6; 7.The drug selected for study is dill oil and clove oil which are effectual oils.8
Along with the essential oils the marigold infused oil is incorporated since the marigold
flowers are the good source of essential oil.9.The marigold infused oil is reported to be
antibacterial, astringent, antifungal, used in insect bites etc10, 11, 12.

MATERIAL AND METHOD

I) Preparation of plant material
The flowers and plants of marigold were collected from a nursery at Vangani village
(Mumbai). The plants were identified and voucher specimens were deposited at the
herbarium of Botany department of Nagpur University. All the chemicals were purchased
from S.R. Traders, Ulhasnagar, and Mumbai. The mosquitoes were collected from Haffkine
Institute for training, Research & Testing, Parel, and Mumbai.

II) Extraction of marigold oil

The collected marigold flowers were introduced into a glass jar. And the glass jar was then
filled with castor oil.The marigold flowers were kept for extraction.
(Infusion) for about 3 weeks. After 3 weeks the Marigold oil was filter and collected into
second jar which was incorporated in the formulation.

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FORMULATION
Infusion of marigold oil was prepared first and was evaluated for repellent activity. The
researchers found significant repellency activity in marigold oil which was compared with
castor oil since the infusion was prepared in castor oil. With the data of marigold oil
repellency activity it was decided to prepare a mosquito repellent cream of different
concentrations i.e. 5% and 7.5% , a combination of marigold oil with two essential oils i.e.
clove oil and dill oil.

Preparation of Mosquito repellent product

Mosquito repellent cream: - An aqueous cream (oil in water type) was prepared by
emulsifying the essential oils in water with an emulsifying wax. The formula for the insect
repellent cream is as follows:
Essential oil 5%or 7. 5%
Cetyl alcohol 2%
Lanolin 1%
Mineral oil 2%
Stearic acid 15%
Glycerin 10%
Pot. Hydroxide 1%
Water Q.S. 100%

EVALUATION OF MOSQUITO REPELLENT ACTIVITY

Mosquito repellent activity was assessed by using the test cage as described in the American
Society for Testing and Materials (ASTM) standard E951-83 Laboratory testing of non-
commercial mosquito repellent formulation on the skin. The test procedure was similar to that
describe by Buescher et al (1982) and Gupta et al (1989) .According to the method 20
mosquitoes were transferred into the cage with the help of aspirator tube. The formulations to
be tested were applied to the forearms of the volunteers. The applied hand was introduced
into the cage and the number of bites was recorded. After every 30 minutes the volunteer
hands were subjected to the cage for 3 minutes and the numbers of mosquito bites were
noted. The test procedure was replicated three times. Same procedure was implemented for
control(blank formulation), marigold oil and castor oil. The Percentage protection provided
by repellent cream can be expressed by the formula (Lillie et al., 1988):
Percent Protection = Bites on control - Bites on treated * 100 Bites on check

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Primary skin irritation study13

Guinea pigs of either sex (300-350) were used for the study. The animals were housed in
standard housing conditions of temperature 27± 20 C and humidity of 60±5 % RH. The
animals were fed with suitable diet and water ad-libidum. Two animals were used for study.
The back of animals was shaven before the study. Five patches of equal area (1cm2 each)
were mark on shaven back. The following preparations were applied.
1. Clean with 5% marigold oil
2. Cream containing 7.5& marigold oil
3. Cream containing combination of volatile oil
4. Cream without active ingredient
5. Control site

Procedure: 0.5 gm cream was applied on marked area, spread uniformly and covered cotton
gauze, which was secured by hypo-allergic adhesive tape. The entire trunk was wrap with
an impervious material for 24 hrs period of exposure. This material aids in maintaining the
test patch in position and retards evaporation of volatile substances. During the test period,
guinea pigs were fed at regular interval. At the end of 24 hrs the patches were remove and the
skin was observed for any visible changes such as erythema or edema. Evaluation was done
by using scale given by Draize. Observation was repeated after 72 hrs.

Draize Scale for the evaluation of the skin reaction

A. Erythema and Eschar formation
a. No erythema 01
b. Very slight erythema (barely perceptible) 1
c. Well define erythema 2
d. Moderate to severe erythema 3
Severe erythema (beet redness) to Slight eschar
e. 4
formation, injuries in depth
Total possible erythema score 4

Oedema formation
a. No oedema formation 0
b. Very slight oedema (barely perceptible) 1
c. Slight oedema (edges of oedema well defined by 2
definite raising)
d. Moderate oedema (area raised approx 1 mm) 3
e. Severe oedema (raised more than 1 mm and 4
expanding behind the area of exposure)
Total possible oedema score 4
Total possible score of primary irritation 8

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pH Determination: The pH of the various gel formulations was determined by using digital
pH meter.
Spredability: It was determined by wooden block and glass apparatus. Weights abut 20gm
were added to the pan and the time as noted for upper slide (movable) to separate completely
from the fixed slide2.

RESULTS AND DISCUSSION

For a repellent to be successful, it must first have to high % protection against mosquito bites.
Second, it should be toxicologically safe at the rate of application for which it is intended.
Third, it should be easy to apply and pleasant on skin.

Formulation parameters
All formulations were subjected to sensory evaluation with respect to color, fragrance,
appearance, shine and ease of applications.All formulations had almost constant pH,
homogeneous, emollient, non-greasy and easily removed after the application. The pH values
of all developed formulations were found to be in the range of 6.7 to 7.

All developed formulations showed good clarity and homogeneity with absence of lumps or
aggregates. Easy spreadability is one of the important characteristics of any topical
preparations as far as patient compliance is concerned. Moreover if the formulation spreads
easily, its application to the area of skin would be more comfortable.

Skin sensitivity study

The skin irritation study is very important as many cosmetics preparation have some inherent
irritation levels. All formulations were safe in respect to skin irritation and allergic
sensitization as the primary irritation index by Draize Patch technique was found to be zero,
and there was no any report of any oedema or redness. Thus indicating skin acceptability of
these formulations for topical application

Mosquito Repellency
Mosquitoes have very olfactory chemoreceptor on sensitive antenne which becomes
stimulated by very distinct odors, it may either increase or decrease attractiveness to the host.
This behavior of reducing the attraction of mosquitoes to their host had been observed during
formulation containing combination of essential oils and marigold oil.

Mosquito repellent activity of extracted marigold oil was checked against Aedes mosquito.

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Based on the results of Marigold oil (59.05% protection) against mosquito bites, it suggested
that the oil has the potential to be developed into formulation for mosquito repellency. Hence,
the extracted marigold oil was incorporated in cream base in different concentration and
evaluated for its activity. The cream containing 7.5% marigold oil provided high level of
protection (56.90%) against mosquito bites. Subjects treated with cream containing 7.5%
marigold oil received only 8 mosquito bites in 90 min. The mean numbers of bits received for
control volunteer were 18.67. Subjects treated with cream containing 5% marigold oil
received only 10.67 mosquito bites in 90 min. The mean number of bits received for control
volunteers were 19.33.This indicate that the cream containing 7.5% provided high
effectiveness.

A cream base containing combination of volatile oil with 5% marigold oil as an active
ingredient had been also tested for mosquito repellent activity. The significance repellency
effect exhibited by the formulation containing combination of volatile oils with % protection
indicated excellent protection.

Table 1 Values of evaluation parameters of developed formulations

Batch Clari Homogenei Spreadability Skin
pH
ty ty g.cm/sec irritation
5 % MO
Clear Good 6.8 6.0 Nil
Formulation
7.5 % MO
Clear Good 6.8 6.5 Nil
Formulation
5 % MO + CO+
6.
DO Clear Good 7.0 Nil
8
Formulation
MO: Marigold oil; CO: Cinnamon oil; DO: Dill oil

Table 2 Evaluation of mosquito repellent activity of developed formulations

Batch Mean Bites
% Mean protection
Test Control
Marigold oil 3±0*(0.008899) 7.67±2.082 59.05±10.14
5 % MO 10.67±2.52*
19.33±1.53 44.12±15.83
Formulation ( 0.003494)
7.5 % MO
8±1.73*(0.003591) 18.67±3.22 56.90±7.02
Formulation
5 % MO + CO+
3.33±2.52*
DO 20±2 82.67±14.52
(0.000425)
Formulation
Values are represented as mean± S.D. of mosquito bites.*indicates the significantly
(p <0.01) different compared to respective control values. (Students unpair T-test)
[MO: Marigold oil; CO: Cinnamon oil; DO: Dill oil]

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Based on the percentage of protection, the formulation containing combination of volatile oils
was found to be the best followed by the formulation containing marigold oil. In case of
marigold oil formulation, the % protection increased with increasing concentration of
marigold oil so dose dependant response was observed.

CONCLUSION
Active ingredients are the focus and responsible factor of all mosquito repellent formulations.
The cream containing 7.5% marigold oil provided high level of protection (56.90%) against
mosquito bites.In case of marigold oil formulation, the % protection increased with
increasing concentration of marigold oil so dose dependant response was observed. Natural
repellents such as herbal essential oils have been employed as alternative compounds for
repelling mosquitoes and other insects. There are reports on the insect repellency from mint,
citronella, basil, thyme, neem, and lemongrass. Although the history of DEET and other
prominent repellents such as dimethyl phthalate is proved, it is accurate to state that the
combination of different essential oils influences repellent performance. At present study, it
confirmed that natural, non-DEET formulation could be used as mosquito repellents.

ACKNOWLEDGEMENT
The authors would like to appreciate the kind cooperation of Dr. Rupali Tasgoankar,
Principal, Yadavrao Tasgoankar Institute of Pharmacy, Bhivpuri , Karjat for their kind
collaboration in this study and providing the laboratory facilities. The author wish to thanks
Dr. Ramaiya Head of depertment Zoonosis , Haffkine Institute for training, Research &
Testing, Parel, Mumbai for providing the mosquitoes. Kind thanks to Dr. Dhomne
Department of Botany, Nagpur University for authentication of the plant.

Funding: Authors are grateful to Mumbai University for providing funds.

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