































Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202107

Research Article

Open Access

1 Department of Pesticides and Toxicology,

University of Gezira, Wad Medani, Sudan.
2 Blue Nile Natural Institute for Communicable

Diseases, University of Gezira, Wad Medani,

Sudan.

Contacts of authors

* To whom correspondence should be
addressed: yousifassad12@gmail.com

Received: September 24, 2020

Accepted: February 7, 2021

Published: March 30, 2021

Citation: Adam EIY , Assad YOHM,
Eltohami MME, Bashir NHH, Abdelrahman
SH, Nour BYM . Natural Anophelin mosquito
repellents and phytochemical analysis of
ethanol and hexane leaf extracts from four plant
species. 2021 Mar 30;4:bs202107

Copyright: © 2021 Adam et al.. This is an
open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

Data Availability Statement: All relevant data are
within the paper and supplementary materials.

Funding: The authors have no support or
funding to report.

Competing interests: The authors declare
that they have no competing interests.

Natural Anophelin mosquito repellents and phytochemical analysis of
ethanol and hexane leaf extracts from four plant species

Emad I. Y. Adam1
>< , Yousif O.H.M. Assad*1

>< , Marwa M. E. Eltohami1 >< ,
Nabil H.H. Bashir1

>< , Samira H. Abdelrahman2
><, Bakri Y. M. Nour2

>< 

Abstract
Currently mosquitoes control is the cornerstone to minimize the rising number of

mosquito borne diseases. There is an urgent need looking for alternatives to the current re-
liance on synthetic insecticides for the vectors control. Application of active toxic agents
from plant extracts as an alternative control strategy was available from ancient times.
These are non-toxic, easily available at affordable prices, biodegradable and show broad-
spectrum target-specific activities against different species of vectors. Natural products
with repellency properties are urgently needed. An insect repellent works by masking
human scent, and a number of natural and synthetic mosquito repellents were studied.
The main aim of this study was to identify the phytochemicals and compare their poten-
tials as mosquito repellents from the ethanol (Et. OH) and hexane (hex) leaf extracts
(LEs) of Ocimum basilicum, Coleus forskohlii, Eucalyptus camaldulensis and Cymbo-
pogon flexuosus under laboratory conditions at 50 and 100% concentrations. Phytochemi-
cal analysis showed that alkaloids, saponins, flavonoids, terpenes, tannins and terpenoids
were present/or absent in the 4 plant-LEs. At 50% concentration of Et. OH extracts,
C. forskohlii exhibited higher repellency potential on Anopheles gambiae with protection
time of 137.3 min, while O. basilicum, E. camaldulensis and Cy. flexuosus registered pro-
tection time of 30.6, 15.3 and 19 min, respectively. At 100% concentration of ethanol-
extracts, C. forskohlii caused the highest protection time against the vector with protection
time of 182 min. O. basilicum, E. camaldulensis and Cy. flexuosus were equally less po-
tent against the insect, with protection time 42.6, 32.6 and 28 min, respectively. Regarding
hex-extract, at 50% concentration of C. forskohlii, the highest repellency potential , with
protection time of 174 was registered, while O. basilicum, E. camaldulensis and Cy. flexu-
osus registered protection times of 44, 18 and 28.6 min, respectively. However, the 100%
concentration of Et. OH-extracts of, C. forskohlii and O. basilicum exhibited the highest
protection time, with protection times of 228 and 116min, respectively. E. camaldulensis
and Cy. flexuosus were less potent, with protection times 28.6 and 54.6 min, respectively.
The EOs of C. forskohlii and O. basilicum L EOs proved to have potentials as repellency
agents against Anopheles. GC-MS analysis of the extracts identified the compounds of
both solvents extracts and the terpens were the major compounds. According to the re-
sults, further studies are required, especially on the extracts of basil and coleus since they
proved to be effective.

Keywords: Mosquito repellent, Anopheles, Essential oils, Sweet Basil, Wild Thyme, Kafure,

Lemon Grass

Introduction
Soonwera and Phasomkusolsil [1] stated that mosquitoes are pestiferous insects to public health;

transmit several deadly dangerous diseases, e.g. dengue (DF), filariasis, malaria, yellow fever (YF)

and Japanese encephalitis (JE). Several researchers reported that every year at least 500 million

people in the world suffer from one of the tropical diseases that include DF, malaria and filariasis [2,

3].

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https://creativecommons.org/licenses/by/4.0/
strongmass2020@gmail.com
yousifassad12@gmail.com
https://orcid.org/0000-0003-2928-3051
marwatoxic@gmail.com
bashirnabilhh@gmail.com
samhamid2002@yhoo.co.uk
https://orcid.org/0000-0001-7714-9266
bakrinour@gmail.com
https://orcid.org/0000-0002-2066-4176
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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Salih et al., [4] reported mosquitoes are among the most
disturbing blood -sucking insects afflicting human beings. The
mosquito female's behavior, involving feeding on human blood,
is responsible for the transmission of a number of diseases. One
to two million deaths is reported annually, due to malaria world-
wide.

Pest/vector management is facing economic and ecological
challenges worldwide, due to human and environmental hazards
caused by majority of the synthetic pesticides. Identification of
novel effective insecticidal compounds is essential to combat
the increasing resistance rates. Botanical pesticides have long
been touted as attractive alternatives to synthetic pesticides for
pest/vector management, because botanicals reputedly pose lit-
tle threat to the environment or to human health. The body of
scientific literature documenting bioactivity of plant derivatives
to arthropod pests/vectors continues to expand, yet only a hand-
ful of botanicals are currently used in agriculture, public health,
industrialized world, and there are few prospects for commercial
development of new botanical products.

The medical vectors control is considered the most impor-
tant corner-stone to prevent the diseases; however, the control is
either by the synthetic pesticides or the repellents that prevent
the biting of the mosquitoes. Personal repellents are substances
applied to skin, clothing, or other surfaces to repel or discourage
insects and other arthropods, e.g. ticks, from feeding on humans.
Repellents help people to avoid bites from mosquitoes, and other
biting arthropods that may transmit diseases, and allow people
to engage freely in outdoor activities. Insect repellents may also
be used to exclude insects from an area [5].

With increasing public concern regarding pesticide safety
and problems of insecticide resistance, new safer active ingredi-
ents are becoming necessary to replace the existing compounds.
Furthermore, the use of repellents in an integrated pest/vector
management (IPM/IVM) program has been ignored to a large
extent [5]. Salih et al., [4] reported that the personal protec-
tion from mosquito bites is currently the most important mea-
sures to control mosquito transmitted diseases. The mosquito
repellent products commonly available on the market contain N,
N-diethyl- 3-toluamide (DEET) as the active ingredient. The
synthetic repellents are not safe for human, especially children,
because they may cause skin irritation, hot sensation rashes or
allergy [6].

Recently, as results of the public health concern on the safety
of many synthetic products, many research scientists renewed
the interest on the use of natural products from plant origin for
mosquito's management [1]. In addition, plant essential oils
(EOs), in general, have been recognized as an important natu-
ral resource of insect repellents. The plant EOs base repellents
according to Soonwera and Phasomkusolsil [1] are effective for
mosquito control, and are environment friendly.

Vector borne disease such as malaria is a major global health
problem; half of the world's populations, mostly the poorest
countries living in the tropical world's are at risk of malaria. The

disease caused by Plasmodium parasites transmitted by infected
female Anopheles mosquitoes bit is serious problem in Africa,
where one in every five (20%) childhood deaths is due to malaria.
Most malaria cases and deaths are in sub-Saharan Africa Most
malaria cases and deaths are in sub-Saharan Africa [7].

Therefore, herbal insect repellents gained importance recently;
detailed knowledge concerning them is still lacking. As a re-
sult, it is high time to launch extensive search to explore eco-
friendly biological/botanical materials for controlling pests. Cur-
tis [8] Choochote et al., [9] reported that EOs have been inves-
tigated and described as potentially natural sources of insect re-
pellents. The effective repellency against female Anopheles re-
sults varied with different species of the plant and the concen-
tration/dose. According to Trongtokit et al., [10] study on An.
stephensi in Brazil using Cymbopogon nardus citronellal 40%
EOs was applied topically, reported 100% protection for 7-8 hr.
The present study investigated the potential of sweet basil (Oci-
mum basilicum L), wild thyme (Coleus forskohlii), Kafure (Eu-
calyptus camaldulensis) and, lemon grass (Cymbopogon flexuo-
sus), LEs as mosquitoes repellents.

Curtis [8] stated in many parts of the world, plant derived nat-
ural products have traditionally been used as repellents against
insects bites. Particularly essential oils have been investigated
and described as potentially natural sources of insect repellents
it is time to search for effective alternatives to synthetic repel-
lents as reported by Choochote et al., [9]. Many research stud-
ies were conducted on various medicinal plants and the effec-
tive repellency against female anopheles mosquitoes, the result
is varying according to the plant and the concentration. The
present study investigates to figure out the effectiveness of Oci-
mum basilicum L., Coleus forskohlii, Eucalyptus camaldulensis
and, Cymbopogon flexuosus Steud, (leafs) extract as mosquitoes
repellent.

Materials and Methods
Collection of plant material and extraction

Fresh leaves of the four above-mentioned plants were col-
lected from Gezira State, Sudan. The collected leaves were
washed, shade-dried and powdered. The powder was extracted
in ethanol (Et. OH) and hexane (hex) by soaking in Et. OH, and
clevenger apparatus was used for preparation of hex- extract; 50
g of each plant leaves powder (LP) was extracted separately in
conical flasks by Et. OH. Analytical grade Et. OH was added
until the LP was fully submerged, thoroughly stirred to ensure
proper mixing, and subjected to shaking at regular intervals to
allow percolation for 4 days. On the 5th day, the extracts were fil-
tered using Whatman No.1 filter paper into another conical flask
and the Et. OH was evaporated. The resultant viscous substance
was dried and stored in amber-colored vials in a refrigerator at
4oC pending formulation. For hex-extracts the EO was isolated
by steam-distillation, using a clevenger apparatus for 6 hr; mois-
ture in the oil was removed by anhydrous sodium sulfate and
stored at 5oC for further use.

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Formulation of plant leaves extracts
The plant leaves extracts to be tested were formulated in

pure fragrance-free Vaseline jelly. The formulations tested were
made into two concentrations of 50% and 100%. To make 50%
of the formulation, 5g of the pure Vaseline jelly was weighed
and transferred to a clean 100 ml beaker, placed in a water bath
at 80°C, stirred with a rod up to the melting point. Five g of each
of the 8 extracts was then added to the melted jelly, followed by
continuous stirring to be completely mixed with the jelly. The
resultant 8 formulations of eight plant extracts were stored at
4°C for the bioassay. To make 100% of the formulation, 1 g of
pure Vaseline jelly was melted as above; 10 g of each of the 8 ex-
tracts were added and treated as above. The two concentrations
(50 and 100%) were used in bioassay.

Anopheline mosquito
The Anopheles gambiae (3-7 days old) was obtained from

Blue Nile National Institute for Communicable Disease (BN-
NICD) insectary, University of Gezira, colony maintained at 12:
12 LD photoperiod, 70±10% R.H. and 24±2oC. The adult fe-
males of Anopheles used for the bioassay before any blood meal.
Females were starved for 24 hr and provided with only 10%
sugar solution in water. Tests were conducted in triplicate 25 in-
dividual of starved adult mosquitoes rearing cage was used and
topical treated arm with plant extract exposed to the mosquitoes
in the cage to observe the mosquitoes bite.

Bioassay test
The reared mosquitoes were taken from the insectary of Blue

Nile Institute for Communicable Disease (BNICD) (24±2Cº and
80% RH) and the experiment was conducted in the insectary lab
of BNICD, the arm cage method was used. Three gram of for-
mulated plant leaves extract was applied onto the forearm of a
human subject. The treated arm was exposed until the first bite
to 25 female mosquitoes, in a (252x3.14x60 cm3) cage, and any
mosquitoes landing and biting were counted. After any bite the
time was recorded at which the first bites occurred the bioassay
terminate and repeated for the other 50 mosquitoes to make trip-
licate at successive time intervals. For the negative and positive
control, the arm with Vaseline jelly and with DEET was used
respectively. Numbers of mosquitoes landing and biting in a
minute were recorded. The application of plant extract was used
following Frances methods [11]. Two concentrations (50 and
100%) for each plant, ethanol and hexane extract and mixture
of ethanol and hexane for each plant to account the number of
mosquitoes land and the complete protection time (CPT) in the
treated area (20-15cm2), with 3g formulated plant extract.

Phytochemical Analysis
The phytochemical analysis was carried out at chemistry lab,

Faculty of Agricultural Sciences, University of Gezira by using
the method of Balbaa [12] and Harborne [13] to investigate the
presence of the saponins, tannins, sterols, triterpens, flavonoids,
terpenoids and alkaloids.

Gas chromatographymass spectrometry
The qualitative and quantitative analysis of the sample was

carried out by using Gas Chromatography-Mass Spectrometer
(GC/MS-QP2010-ultra) from japans Simadzu company, with the
serial number 020525101565SA and capillary column (Rtx-5ms-
30m[2E3?]0.25mn[2E3?]0.25μm).The sample was injected by
using split mode, Helium as A carrier gas passed with flow rate
1.61 ml/min, the temperature program was started from 60c with
rate 10oC/min to 300oC as a final temperature degree with 5min-
utes hold time, the injection port temperature was 300oC as the
ion source temperature was 200oC and the interface temperature
was 250oC. The sample was analyzed by using scan mode in
the range of m/z 40-500 charges to ratio and the total run time
was 29 minutes. Identification of the components for the sample
was achieved by comparing their retention index and mass frag-
mentation patents with those available in the library, the national
institute of the standards and technology (NIST).

Data Analysis
For each plant species, the number of mosquitoes landed on

the arm was counted at different time intervals and the Percent-
age Repellency (PR) per each plants leaf extract/solvent/concentration
was determined using the following equation (1) according to
Teklani and Perera [14] as follows:

PR =
NIU − NIT

25 (IS )
100 (1)

Where:

• PR=Percentage repellency.
• NIU=Number of individual in the untreated group.
• NIT=Number of individual in the treated group.
• IS=Number of insects used.

At least triplicates were carried out for all the plants leaves
extracts for selected extracts concentration with significant mosquito
repellent activity.

Results
Phytochemical analysis

Phytochemical analysis of ethanolic and hexane extracts of
the four plants leaves were carried out to identify the presence
of Alkaloid, Tannin, Saponin, Terpenoids, Flavonoids and ter-
pens. In this study four plant species (Basil, Wild Thyme, Ka-
fure and Lemon Grass) belonging to different families were col-
lected. Most of these plants were reported to treat a variety of
diseases in traditional medicine system.

Ethanol Extracts
The preliminary phytochemical screening had shown the pres-

ence of Alkaloid, Tannin, Flavonoids, Saponin and Terpens in
the leaves extracts the summary of the results are presented in
Table 1. The Alkaloid, Tannins, saponin and Terpens were de-
tected in the Basil leaves extract; Alkaloid, Tannins, and Terpens
in Wild Thyme leaves extract; Alkaloid, Tannins, Flavonoids
and saponins in Kafure leaf extract and Alkaloid,Tannins, and
saponins in Lemongrass,

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Table 1. The phytochemical constituents (PC)of ethanol (E) and hexane (H)
leaf extracts from the studied four plants.

PC Oc Col Eu Cy

E H E H E H E H

Alkaloid + + + - + + + +

Tannins + + + - + + + +

Flavonoids - - - - + + - -

saponins + - - - + + + +

Terpens + - + + - - - -

Oc=Ocimum, Col=Coleus, Eu=Eucalyptus, Cy= Cymbopogon.

(+)= Presence, (-) = Absence

Hexane extract
The preliminary phytochemical screening had shown the pres-

ence of Alkaloid, Tannin, Flavonoids, Saponin and Terpens in
the hexane leaves extracts the summary of the results are pre-
sented in Table 1. In Basil leaves extract Alkaloid, Tannins
was only detected. While in Wild Thyme leaves extract terpens
only was detected. In Kafure leaves extract Alkaloid, Tannins,
Flavonoids and Saponins were detected and Terpens is absent.
In Lemongrass extract Alkaloid, Tannins, and Saponins were de-
tected.

Bioassay
The results of bioassay were collected as a complete protec-

tion time (CPT), and the number of mosquitoes landing for the
concentrations (50 and 100%), ethanol, hexane and hexane plus
ethanol extract. Ethanol extracts presented in Tables 2 and 3
shows that Wild Thyme 50% and 100% were recorded the high-
est protection time 137.3 and 182 min respectively compare to
the other plant extracts, with percent repellency 92 and 89% re-
spectively. For the hexane extracts presented in Tables 4 and
5 prove that Thyme 50% and 100% were recorded the highest
protection time 124 and 228 min respectively compare to the
other plant extracts, with percent repellency 90 and 92% respec-
tively. Regarding the combination of the two extracts presented
in Tables 6 and 7 also the Wild Thyme 50% and 100% were
recorded the highest protection time 182 and 215 min respec-
tively compare to the other plant extracts except Basil in 50%
recorded higher protection time (86 min), with percent repel-
lency 84 and 88% respectively. By the way the selected four
leaf plant extracts offers protection against An. arabiensis adult
female mosquitoes but varied in terms of repellency up to 228
minutes of exposure periods recorded by Wild Thyme.

Chemical constituents detected in Kafure leaf extracts
Table 8 shows the chemical constituent of Kafure leaves

etha- nol and hexane extracts; their concentrations, peak area,
retention time and chemical group of each compound. A total
of 13 compounds detected in ethanol and hexane extract which
represent 63.63% and 68.54% for ethanol and hexane extract,
respectively. Eucalyptol was the main compounds (33.11% and
26.93%), respectively. Table 9 shows the chemical constituent

of Kafure leaf's ethanol extract; their concentrations, peak area,
retention time and chemical group of each compound. A total of
20 compounds detected in ethanol extract and represent 36.37%
of the total of the extract. Globulol 9.94% and Squalene 8.10%
were the main compounds. Table 10 shows the chemical con-
stituent detected in Kafure leaf's hexane extract; their concen-
trations, peak area, retention time and chemical group of each
compound. A total of 25 compounds detected in hexane extract,
as follows, where Globulol was the main compound (18.06%).

Discussion
The synthetic chemicals used to control insects leads to sev-

eral documented environment and human health hazards. An
alternatives method suggested using natural products that pos-
sess good efficacious results and environmental friendly. Among
those alternative methods the use of essential oils from plants
belonging to several plant species subjected intensive studies
which provide successful results against field crop pests and pub-
lic health pests and disease vectors control, including mosquitoes
(Anopheles, Culex and Aedes). These EOs were extensively
tested by several scientists for their repellent properties as valu-
able natural resources and to elucidate their chemical and physi-
cal properties, in addition to their modes and sites of action, for-
mulation, toxicological profiles, side-effects, etc... Today medi-
cal vector management has to face environmental and economic
costs.

Table 2. Spatial repellent activity of 50% and 100% evaluated plants leaves
ethanol extract against Anopheles mosquito based on the number of landed
mosquitoes (NLM) and protection time per minute (PT).

Treatment 50% 100%

NLM PT NLM PT

1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean

Control 25 24 24 24.33 - - - - 25 24 24 24.33 - - - -

DEET 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66

Wild Thyme 1 2 1 1.3 113 122 177 137.3 2 3 1 2 210 154 182 182

Sweet Basil 2 3 1 2 25 36 31 30.6 4 2 6 4 42 31 45 42.6

Kafure 4 2 1 2.3 15 10 21 15.3 2 3 4 3 25 32 41 32.6

Lemongrass 2 3 5 3.3 15 17 25 19 4 2 3 3 31 25 28 28

Table 3. Percent repellency (PR) of 50% and 100% of evaluated plants leaves
ethanol extract against Anopheles mosquito based on the number of landed
mosquitoes (NLM).

Treatment 50% 100%

NLM PR NLM PR

1st 2nd 3rd mean 1st 2nd 3rd mean

Control 25 24 24 24.33 - 25 24 24 24.33 -

DEET 8 9 5 7.33 68% 8 9 5 7.33 68%

Wild Thyme 1 2 1 1.3 92% 2 3 1 2 89%

Sweet Basil 2 3 1 2 89% 4 2 6 4 81%

Kafure 4 2 1 2.3 88% 2 3 4 3 85%

Lemongrass 2 3 5 3.3 84% 4 2 3 3 85%

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Table 4. Spatial repellent activity of 50% and 100% evaluated plants leaves hexane extract against Anopheles mosquito based on the number of landed
mosquitoes (NLM) and protection time per minute (PT).

Treatment 50% 100%

NLM PT NLM PT

1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean

Control 25 24 24 24.33 - - - - 25 24 24 24.33 - - - -

DEET 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66

Wild Thyme 1 2 2 1.6 191 142 189 174 1 2 1 1.3 230 208 246 228

Sweet Basil 3 2 5 3.3 46 36 50 44 2 1 3 2 90 118 140 116

Kafure 4 6 8 6 18 21 15 18 3 1 2 2 36 21 28 28.3

Lemongrass 3 4 6 4.3 22 36 28 28.6 2 3 5 3.3 59 41 63 54.3

Table 5. Percent repellency according to the number of landed mosquitoes (NLM) of 50% and 100% percent of evaluated plants leaves hexane extract against
Anopheles mosquito.

Treatment 50% 100%

NLM PR NLM PR

1st 2nd 3rd mean 1st 2nd 3rd mean

Untreatedcontrol 25 24 24 24.33 - 25 24 24 24.33 -

DEET 8 9 5 7.33 68% 8 9 5 7.33 68%

WildThyme 1 2 2 1.6 90% 1 2 1 1.3 92%

SweetBasil 3 2 5 3.3 84% 2 1 3 2 89%

Kafure 4 6 8 6 73% 3 1 2 2 89%

Lemongrass 3 4 6 4.3 80% 2 3 5 3.3 84%

Table 6. Spatial repellent activity and protection time per minute (PT)of 50 and 100% of evaluated plants leaves hexane and ethanol extract mixture against
Anopheles mosquito.

Treatment 50% 100%

NLM PT NLM PT

1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean

Control 25 24 24 24.33 - - - - 25 24 24 24.33 - - - -

DEET 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66

Wild Thyme 2 3 5 3.3 164 194 190 182.6 4 2 1 2.3 211 233 201 215

Sweet Basil 4 1 3 2.6 48 51 31 43.3 2 6 4 4 66 94 101 87

Kafure 4 6 7 5.6 17 11 19 15.6 6 5 8 6.3 20 15 23 19.3

Lemongrass 6 3 4 4.3 15 24 19 19.3 1 2 4 3.3 27 20 31 26

Table 7. Percent repellency (PR) according to the number of landed
mosquitoes (NLM) of 50% and 100% of evaluated plant leaves hexane and
ethanol extracts mixture against Anopheles mosquito.

Treatment 50% 100%

NLM PR NLM PR

1st 2nd 3rd mean 1st 2nd 3rd mean

DEET 8 9 5 7.33 68% 8 9 5 7.33 68%

Wild Thyme 2 3 5 3.3 84% 4 2 1 2.3 88%

Sweet Basil 4 1 3 2.6 86% 2 6 4 4 81%

Kafure 4 6 7 5.6 74% 6 5 8 6.3 72%

Lemongrass 6 3 4 4.3 80% 1 2 4 3.3 84%

The Synthetic repellents DEET and pyrethroids containing
formulations commonly sold in markets and insecticides sales
vender. The side effects of these two chemical groups was re-
ported by several research scientists, accordingly plant-based re-

pellents not only minimize the use of synthetic chemicals and
economic cost and save huge amount of money spent on syn-
thetic compounds, also protects the environment, health of users.
The plants screened for phytochemical analysis seemed to have
the potential to act as a source of useful chemicals and also to
improve and help in the health status of the people exposed to
mosquito's bits (Table 1). This study justifies the use of plant
species in medicinal field to protects human from mosquito's bits
and hence minimize the malaria spreading. There are different
plants which have Mosquito repellent activity and this activity
affected with species the environment and other factors. And
the chemical constituent of these show different results on the
mosquito repellent. The different plant extract contains differ-
ent chemical constituent shows different results on the mosquito
repellent activities (Tables 2-7).

This study investigated the mosquito spatial repellent effi-

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

cacy of the basil, wild thyme, kafure and lemongrass, (ethanol,
hexane and mixture of both ethanol extract and hexane extract).
The present study results provided information and observations
on the repellency activity of tested four plant species LEs against
Anopheles mosquitoes. Mosquitoes usually make surreptitious
landings on exposed skin to feed this why most people wearing
protective clothing stands as a good measure of preventing them
from vector contact. Application of these plants LEs prevented
landing attempts and bites by mosquitoes; this verifies already
documented evidence that some plants LE is a potent mosquito
repellent.

Sweet basil
O. basilicum EOs are materials that are traditionally used

in Sudan the mosquitoes by burning leafs and flowers. How-
ever, Coker et al., [15] stated that certain characteristics, e.g.
volatility, limited its effectiveness. Active ingredients, e.g. ter-
penes (2◦ metabolites) and oil components are responsible for
the repellency activity. For a plants material to be valuable as a
mosquito repellent it must effectively prevent insect bites on the
treated area for several hours, and it must work in different envi-
ronmental conductions, should be environmental- friendly when
applied to human skin, it must acceptable and having a pleasant
odor, it should also be harmless to clothing, it should have a rel-
atively low cost and effective against other biting insects such
as flies. Basil leaves extract provide different spatial repellency
against mosquitoes with different concentration, in ethanol, hex-
ane and mixture of ethanol and hexane extract (Tables 2, 4 and
6). Ethanol extract provided 30.6 and 42.6 min 32 and 45%
for 50% and 100% ethanol extract respectively and the hexane
extract was provided 44 and 116 min and evaluated with 46%
and 122% for 50% and 100% respectively when compared with
DEET, while the mixture of ethanol and hexane extract shows
that basil was provides the protection time in both (50% and
100%) was 43 min and 87 min respectively and this repellency
act as 45% and 91.9% when compared with standard mosquito
repellent DEET (94.6 min protection time) respectively (Tables
3, 5 and 7). Basil hexane leaves extract provide the highest repel-
lency against mosquitoes (116 min), Baba et al., [16] conclude
that hexane basil leaves extract provided 183 min and 303 min
CPT from concentration 50% and 100% respectively (Tables 4
and 6). In this study; the presence of ethanol extract in the mix-
ture was reduced the repellency of mixture from the 116 to 87
min in 100% concentration; so the tannin and alkaloids may re-
sponsible for the repellent activity in hexane extract according to
the phytochemical analysis (Table 1). The phytochemical anal-
ysis was preceded an Alkaloids, Tannins, saponins and terpens
were found in the Ethanol Extract, While Alkaloid and Tannins
only detected in Hexane extract..

Wild thyme
Ethanol extract, the wild thyme was provides the highest pro-

tection time in both (50 and 100) %137.3 min and 182 min re-
spectively when compared with other plant ethanol extract, and

repellency at 154% and 192%, respectively when compared with
standard mosquito repellent-DEET (94.6 min protection time).
Active movement away from treated arm within minute of appli-
cation was observed in wild thyme, and this strong repellent ac-
tivity may caused by terpens group which appear in ethanol and
hexane extract (according to phytochemical analysis) as main
compounds and thymol (monoterpenoid) was highest concentra-
tion (54.96% and 57.42%) in ethanol and hexane extract respec-
tively according to GC-MS analysis, and this was confirm by the
study done by Pandey et al., [17] they prove the activity of thy-
mol that repel An. stephensi adults at the dose of 25.0 mg/mat
after 1 h duration, while same degree of repellency was obtained
by the oil at the dose of 55.0 mg/mat, which indicating thymol
has double activity than the essential oil obtained from seeds of
Trachyspermum ammi (Linn.). In present study beside the alka-
loids and tannins present in ethanol extract only (phytochemical
analysis), the hexane extract of the wild thyme is provides the
highest protection time in both (50 and 100) % 174 min and
228 min respectively when compared with other plant leaves ex-
tracts and this highest CPT and repellency as 183% and 241%
respectively, when compared with standard mosquito repellent-
DEET (94.6 min CPT). Regarding the mixture of ethanol and
hexane extract of each of the tested plants, wild thyme is pro-
vides the highest protection time in both (50 and 100%) 182.3
min and 215 min with spatial repellency at 193% and 227% re-
spectively, compared to DEET. The main compounds detected
in ethanol and hexane extract was thymol which shows consid-
erable anti-feedant activities against insects in many studies con-
ducted by Gonzalo et al., [18] and Won et al., [19] shows that the
wild thyme essential oil was rich source monoterpenes, includ-
ing thymol, p-cymene, carvacrol, linalool, and o-terpinene, were
assessed to determine their repellent activities to the mosquitoes.
o-Terpinene had a potent spatial repellent activity with a protec-
tion rate of essential oil topical treatment while the carvacrol
and thymol showed an equivalent level of repellency. The Wild
thyme was the best one compared with the rest plants it was
provided highest CPT; in both ethanol and hexane extract. How-
ever, the hexane 100% was provided the protection until 228
min, this period even longer than DEET (2.4 times). According
to phytochemical analysis the spatial repellency of Wild thyme
could be rise from terpens mainly because the hexane extract
shows the terpens only. although provided the highest CPT and
the ethanol extract which contain terpens beside the alkaloids
and tannins show low CPT. Our study show that thymol and eu-
calyptol (monoterpenoid) was main compounds in either ethanol
and hexane extract and its concentration was corresponding with
spatial repellency period, the repellency of plant extract could
be due to the eucalyptol and this was proved by James et al.,
[20] they found that mosquito feeding and ovipositional repel-
lency due to the presence of major monoterpenoid in the chem-
ical volatile oil (i.e., 1,8-cineole (eucalyptol) identified in plant
extract of Hemizonia fitchii (Asteraceae).

Phytochemicals are the principal active components that are

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Table 8. Chemical constituent detected appear in the GC-Mass chromatogram in both ethanol and hexane extract of Kafure leaf’s and area percentage of peaks.

ID Name Area% Ethanol Area% Hexane

1. Alpha-Pinene 3.38 6.77

2. D-Limonene 0.19 0.55

3. Eucalyptol 33.11 26.93

4. Gamma.-Terpinene 0.30 0.47

5. Pinocarvone 1.06 1.94

6. 2-Oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl-, acetate 0.25 0.37

7. Bicyclo(3.1.1)heptane-2,3-diol, 2,6,6-trimethyl- 0.18 0.24

8. Copaene 0.19 0.42

9. Alloaromadendrene 0.39 0.26

10. Caryophyllene 0.76 0.92

11. Aromandendrene 7.02 7.07

12. Alpha.-Guaiene 1.60 2.37

13. 2-Naphthalenemethanol, decahydro-.alpha.,.alpha.,4a-trimethyl-8-methylene-, [2R-(2.alpha.,4a.alpha.,8a.beta.)] 15.20 20.23

Table 9. Chemical constituent detected appear in the GC-Mass chromatogram in ethanol extract of Kafure leaves and area percentage of peaks.

ID Name Peak Area(%)

1. alpha-Phellandrene 0.79

2. p-Cymene 1.78

3. 3-Methyl-4-cyclohexene-1,2-dicarboxylic anhydride 0.20

4. Benzene, 1-methyl-4-(1-methylethenyl)- 0.67

5. Isoledene 0.11

6. 1-Tridecene 0.12

7. Bicyclo[2.2.2]octa-2,5-diene, 1,2,3,6-tetramethyl- 0.15

8. Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]- 0.04

9. Naphthalene, decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-, [4aR-(4a.alpha.,7.alpha.,8a.beta.)]- 0.18

10. 1H-Cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1aR-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.)- 0.21

11. (S)-(-)-4-Isopropenyl-1-cyclohexene-1-carboxylic acid 1.98

12. Azulene, 1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1R-(1.alpha.,3a.beta.,4.alpha.,7.beta.)]- 1.42

13. Guaia-1(10),11-diene 1.37

14. 7-epi-cis-sesquisabinene hydrate 0.79

15. Globulol 9.94

16. Phytol, acetate 3.03

17. 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 0.55

18. 9-Eicosyne 1.04

19. n-Hexadecanoic acid 3.90

20. Squalene 8.10

believed to exhibit the medicinal activity of the plants and pos-
sibly the repellent activity of the oils. According to Banthorpe
[21] and Heinrich et al., [22] terpenoids could be responsible for
the flavor of fruits, the fragrance of the flowers and the quality of
agricultural products. Therefore Coker et al., [15] prove that the
presence of terpens is speculated to be associated with fragrance
material and spatial repellent activity of oils.

Kafure
Kafure leaves extract provided different CPT by (50% and

100%) concentration, in ethanol; hexane and ethanol and hexane
extract (mixture) (Tables 2, 4 and 6). Ethanol extract provide 15
min and 32 min by 50% and 100% concentration respectively.
However the hexane extract provide 18 min and 28 min com-

plete protection by 50% and 100% concentration respectively
when the mixture provided 15 min and 19 min complete pro-
tection by 50% and 100% concentration respectively. The result
shows that hexane extract was slightly stronger repellent than the
ethanol and the mixture; the phytochemical analysis shows the
presence of the: saponins, tannins, and alkaloids and absence of
triterpens in both ethanol and hexane extract; while the GC-MS
detected some of monoterpenes like euclyptol, (Cyclic) 33.11%
and 26.93% and 2-Naphthalenemethanol, decahydro-alpha, al-
pha, 4a-trimethyl-8-methylene-, [2R-(2alpha, 4aalpha, 8abeta )]-
15.20% and 20.23%, in ethanol and hexane extract respectively,
and Globulol was 9.94% in ethanol extract and hexane extract
was contained (-)-Globulol 18.06% so this variety may respon-

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

Table 10. Chemical constituents detected appear in the GC-Mass chromatogram in hexane extract of Kafure leaves and area percentage of peaks.

ID Name Peak Area(%)

1. Camphene 0.14

2. Bicyclo[3.1.0]hex-2-ene, 4-methylene-1-(1-methylethyl)- 0.07

3. beta-Pinene 0.22

4. beta-Myrcene 0.06

5. o-Cymene 3.58

6. Doconexent 0.20

7. Butanoic acid, 3-methyl-, 3-methylbutyl ester 0.28

8. alpha-Campholenal 0.14

9. Terpinen-4-ol 1.58

10. L-.alpha.-Terpineol 1.67

11. Ethanone, 1-(6,6-dimethylbicyclo[3.1.0]hex-2-en-2-yl)- 0.19

12. 2-Cyclohexen-1-one, 3-methyl-6-(1-methylethyl)- 0.08

13. Citral 0.15

14. 1H-Cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1aR-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.) 0.20

15. 12-Oxatetracyclo[4.3.1.1(2,5).1(4,10)]dodecane, 11-isopropylidene- 0.17

16. Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2R-(2.alpha.,4a.alpha.,8a.beta.)]- 1.38

17. beta-Humulene 0.31

18. 2H-3,9a-Methano-1-benzoxepin, octahydro-2,2,5a,9-tetramethyl-, [3R-(3.alpha.,5a.alpha.,9.alpha.,9a.alpha.)]- 0.22

19. gamma-Muurolene 0.21

20. Aromadendrene, dehydro- 0.20

21. 2-Oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl- 0.26

22. 1H-Cycloprop[e]azulen-4-ol, decahydro-1,1,4,7-tetramethyl-, [1aR-(1a.alpha.,4.beta.,4a.beta.,7.alpha.,7a.beta.,7b.alpha.)]- 1.32

23. (-)-Globulol 18.06

24. Caryophyllene 0.47

25. 1H-Benzocycloheptene, 2,4a,5,6,7,8,9,9a-octahydro-3,5,5-trimethyl-9-methylene-, (4aS-cis)- 0.27

sible of different repellency and the 32 min was highest CPT
achieved by kafure ethanol extract. In the study conducted by
Fathelrahman [23] showed ethanol extract 10% concentration
provide 17% repellency for 60min. the result was correspond-
ing with this study that increasing extract concentration of ka-
fure was increase the repellency in both extract and the kafure
extract, although was weak in spatial repellency.

According to the result of GC-MS analysis of the chemical
compound of kafure leaves extract of the current study (Tables
8, 9 and 10); the results were corresponding with the GC-MS
analysis of essential oil yield and composition in Eucalyptus
species in Montenegro coastline by Slavenko, et al; [24] show
the presence of α-pinene, β-pinene, β-myrcene, α-phellandrene,
α-terpinene, p-Cymene, Eucalyptol γ-terpinene, terpinene-4-ol,
α-terpineol, aromadendrene, alloaromadendrene, also was found
in this study.

According to the GC-MS analysis of the present study prove
the presence of o-Cymene 3.58%, Terpinen-4-ol 1.58%, L-.alpha.-
Terpineol1.67%, Naphthalene, 1, 2, 3, 4, 4a, 5, 6, 8a-octahydro-
4a, 8-dimethyl-2-(1-methylethenyl), L-alpha -Terpineol 1.67%
[2R-(2.alpha.,4a.alpha.,8a.beta.)]-1.38%, 1H-Cycloprop[e] azu-
len-4-ol,decahydro-1,1,4,7-tetramethyl,[1a R (1a alpha, 4beta,
4a beta, 7alpha, 7a beta, 7b alpha)]-1.32%, (-)-Globulol 18.06%,
2-Naph- thalenemethanol, decahydro -alpha, alpha, 4a-trimethyl
-8-methy- lene-,[2R-(2alpha,4a alpha, 8a beta)]- 20.23%, was

found in the hexane leaves extract and not found in ethanol ex-
tract, and alpha-Pinene was 6.77% in hexane extract and 3.38%
in ethanol extract.

Lemongrass
Application of Cymbopogon oil prevented landing attempts

and bites by mosquitoes; and this verifies already documented
evidence by Baldacchino et al., [25] and Prabhakar et al., [26]
that Cy. Citrates extract is a potent mosquito repellent. However,
the interval of protection is a matter of concern; in this study,
lemon grass leaf extracts provided protection from mosquito for
3 hours after which landing attempts and bites were experienced.
This observation corroborates previous records of plants essen-
tial oils by Bhupen et al., [27].

According to Bhupen et al., [27] has demonstrated the essen-
tial oils from a variety of plant species been used as insect repel-
lents; such as Cymbopogon. In the present study Lemongrass
leaves extract provided different CPT with different concentra-
tion (50% and 100%) in ethanol, hexane and ethanol and hexane
extract (mixture). Ethanol extract provided 19 min and 28 min
by 50% and 100% concentration respectively (Tables 2 and 4).
However the hexane extract provide 28 min and 54 min com-
plete protection by 50% and 100% concentration respectively
(Table 6). While the mixture provided 19 min and 26 min com-
plete protection by 50% and 100% concentration respectively.

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Adam et al., 2021 Natural Anophelin mosquito repellent analysis of leaf extracts of four plant species

The 54 min was maximum CPT provided by lemon grass (hex-
ane extract) with spatial repellency is equal 57% compared with
DEET, and also was low when compared with wild thyme and
basil extract. In phytochemical studies both ethanol and hexane
extract was showed the Alkaloid, tannins and saponins while the
terpens and flavonoids were absent and those groups were impor-
tant as insect repellent. Although monoterpenes was detected by
the GC-MS as main compounds as 2,6-Octadienal, 3,7-dimethyl-
, (Z)-in ethanol extract and 2,6-Octadienal, 3,7-dimethyl-, (Z)-
and 2,6-Octadienal, 3,7-dimethyl-, (E)- in hexane extract. In the
study conducted by Vinutha et al., (28) on essential oil yield
from aerial part of Cymbopogon flexuosus; Myrcenol, Linalool,
Trans-chrysanthemal, 3,6,6-Trimethyl -cyclohex-2-enol, Citronel-
lal, (-)-Isopinocampheol, 1-Pentanol, 5-cyclopropylidene-3- un-
decyne, 3-carvomenthenone, (Z)-linalool oxide, (furanoid), Neral,
Geranial, β-Vatirenene, Citronellol, Dextro-carvone, Cycloiso-
longifolene, Trans-(-)-Carveol, cis-Carveol, Nerol, Oxiranmethan-
ol, 3-methyl-3(4-mathyl-3-pentenyl), Bicyclopentylone, Geranic
acid, Geranyl acetate, most of those compounds were detected
in lemongrass extract in this study.

According to the result, undiluted hexane leaves extract (
100% ) provide maximum CPT according to the follows: 228
min wild thyme, 116 min basil, 54 min Lemongrass and 28 min
kafure, thus the hexane extract was seem more stronger in repel-
lency than the ethanolic extracts, and the Coleus is the best and
effective as mosquitoes repellent than other plants, followed by
Basil and Lemon grass and kafure.

The results of the current work indicated that the spatial re-
pellent potential of the leaves extract oils is concentration depen-
dent. However it is observed that all the thyme leaves extract oils
indicated protection of at least 2.5 h at 50% concentration and
2.5-5 h protection at 100%. Accordingly the use of plants leaves
extract essential oil in prevent mosquito bite is expected to re-
duce the cost and environmental effects of mosquito repellents.

According to Annamaria et al., [29] showed volatile com-
pounds detected in Bixa orellana samples the major volatile com-
pound present in seed extracts proved to be source of repellent
activity (mainly of sesquiterpenes, monoterpenes, arenes and as
α-humulene). These volatile compound found in most of our
chemical analysis to the four plants which reflects the repellent
activity of the test extract especially thyme. In conclusion the
preliminary findings of this laboratory evaluation bioassay of
the repellent potential of the essential oils of Ocimum basilicum
L., Coleus forskohlii, Eucalyptus camaldulensis and, Cymbo-
pogon flexuosus Steud, leaves extracts have confirmed their tra-
ditional use as a broad mosquito repellent agent. Further investi-
gations leading to identification of the lead compounds exhibit-
ing the repellent activity is recommended. This would enhance
a robust development of plant based oil for protection against
mosquito bite. Basil provided considerable repellency against
Mosquitoes. Wild thyme was most effective which provided 105
min CPT evaluated with 205% in compare to the control (DEET,
lavender). The examined plants will be useful if the further

studies have been conducted and it will replace the commercial
mosquitoes repellent (DEET, lavender). Terpenoids were found
in most of the extracts, but in the C. forskohli with the highest
concentration. The presence of the (+)-4-carene, benzene, 1-
methyl-3-(1-methylethyl)-, 2-naphthalenemethanol, decahydro-
were related with hex- extracts of C. forskohli only, and it has
strong repellency activity.

Acknowledgements
We appreciate the assistance from Blue Nile Institute for

communicable diseases for providing the mosquitoes for the stud-
ies. We thank Ministry of Higher Education and Scientific Research-
Commission of Scientific Research and Innovation in supporting
the research fund and thanks to all colleagues helping in a way
or another to let this work done.

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	Abstract
	Introduction
	Materials and Methods
	Collection of plant material and extraction
	Formulation of plant leaves extracts
	Anopheline mosquito
	Bioassay test
	Phytochemical Analysis
	Gas chromatography–mass spectrometry
	Data Analysis

	Results
	Phytochemical analysis
	Ethanol Extracts
	Hexane extract
	Bioassay
	Chemical constituents detected in Kafure leaf extracts

	Discussion
	Sweet basil
	Wild thyme
	Kafure
	Lemongrass

	Acknowledgements
	References

