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Tithonia
diversifolia
Anopheles
coluzzii
*
Tithonia diversifolia
Anopheles coluzzii
T. diversifolia
Cymbopogon (C.) citratus
T. diversifolia
T. diversifolia
T. diversifolia
C. citratus
Aoluzzii
T. diversifolia
T. diversifoliaA. coluzzii.
Abbreviations
A. Anopheles
ACT Artemisinin-based combination therapy
DEET N,N-Diethyl-meta-toluamide
EO Essential oil
EO Essential oil
1Department of Pharmaceutical Sciences, Inorganic Chemistry Lab, Faculty of Medicine and Pharmaceutical
Sciences, The University of Douala, P.O. Box 2701, Douala, Cameroon. 2Department of Animal Biology, Faculty
of Science, The University of Douala, P.O. Box 24157, Douala, Cameroon. 3Department of Biological Sciences,
Parasitology Lab, Faculty of Medicine and Pharmaceutical Sciences, The University of Douala, P.O. Box 2701,
Douala, Cameroon. 4Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC),
Research Institut of Yaoundé, 288 Yaoundé, Cameroon. 5Malaria Research Unit, Centre Pasteur Cameroon,
P.O. Box 1274, Yaoundé, Cameroon. 6Laboratory of Parasitology, Mycology and Virology, Postgraduate Training
Unit for Health Sciences, Postgraduate School for Pure and Applied Sciences, University of Douala, P.O Box 24157,
Douala, Cameroon. *email: elsecarole@yahoo.fr; eboumbou@pasteur-yaounde.org
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FMPS Faculty of Medicine and Pharmaceutical Sciences
GC–MS Gas chromatography–mass spectrometry
LLIN Long-lasting insecticide-treated net
RI Retention index
WHO World Health Organization
Anopheles coluzzii (Diptera: Culicidae) is one of the main mosquito species responsible for malaria transmission
in endemic countries1. Malaria is an infectious disease caused in humans by protozoan parasites belonging to
the genus Plasmodium, Plasmodium falciparum (P.f) and P. vivax being the two species accounting for the bulk
of malaria burden2. e disease is an important public health concern with nearly 229 million cases and 409,000
deaths worldwide, especially in children aged under 5 years and pregnant women3. Malaria burden has been
dramatically reduced over the past two decades. Between 2000 and 2019, malaria case incidence rates dropped
from 362.8 to 225.2 cases per 1000 population at risk. e same pattern was observed for malaria mortality rate
which has fallen from 121.1 to 40.3 deaths per 100,000 population at risk during the same period3.
ese main achievements were the result of implementation and/or scale up of varied malaria control inter-
ventions viz. free distribution of long-lasting insecticide treated nets (LLINs), more reliable diagnosis of the
infection through immunochromatographic rapid diagnostic tests, and their treatment with artemisinin-based
combination therapies (ACTs)3.
Unfortunately, malaria burden is still dramatically high in several endemic countries, especially in sub-
Saharan Africa which bears > 90% of global morbidity and mortality cases3. e emergence and spread of ACT-
resistant P. falciparum populations and insecticide-resistant Anopheles mosquitoes hindered enormous eorts
made in malaria control and elimination4,5. Additionally, LLIN-based mosquito control in African countries is
compromised by the change in biting behavior of Anopheles vectors which increasingly choose to bite humans
indoors in the early evening or bite outdoor (also known as behavioral resistance), thereby limiting the positive
impact of LLINs6–8.
Malaria is a cause of concern in Cameroon with a prevalence of 24% in children under ve, and accounting
for 25.8% of all medical consultations in 20189. Cameroon is also targeted by the WHO “High burden to High
impact” strategy which aims at reducing malaria transmission in 11 countries where malaria burden is highest10.
LLINs are a key control for curbing malaria in the country. e government of Cameroon also encourages the
use of additional personal protection measures, including coils, indoor residual sprays, as well as body repellents,
all of which are widely used by local populations11–13.
Repellents reduce malaria transmission by minimizing mosquito-human contacts. e most commercially
available repellent formulations are either synthetic (e.g., N,N-Diethyl-meta-toluamide-DEET) or derived from
plant extracts (e.g. Neem, Citronella, fennel or Pyrethrum grasses)14. DEET is the oldest and most eective insect
repellent available on the market, but several studies have reported occasional mild-to-severe toxicity reactions
following the application of this molecule on the skin15. Although the above mentioned plant extract-based
repellents are less toxic than their DEET-based counterparts, they are less eective16,17. Similarly, the eective-
ness of Cymbopogon (C.) citratus, Citrus limon, Melissa ocinalis essential oils (EO) has been proven in some
previous studies, albeit with dierences in sensitivity according to vector species18,19. In addition, the duration
of protection of a repellent varies between individuals depending on the amount of lactic acid excreted in the
sweat and other factors of attraction in humans20,21. erefore, more eective and durable repellents are needed
to overcome the current challenges facing this key vector control strategy.
In this regard, EOs represent suitable alternatives for repellent development as they are inexpensive, relatively
safe and durability under body temperature conditions22. In the present study, we evaluated the repellent activity
of T. diversifolia EOs against Anopheles coluzzii mosquitoes. is plant, native to Central America, is now widely
distributed in Australia, Asia and Africa23. Some studies showed an interesting repellent activity of T. diversifolia
EO and aqueous extract (AE) against Anopheles gambiae s.l.24,25, ticks21 and against eas26 and were limited to
West, East Africa and Asia. To the best of our knowledge, no published study evaluated its repellent activity
against A. coluzzii that is widely distributed in Africa.
e leaves of T. diversifolia were collected in Feb-
ruary 2019 from natural habitat in the Fokoue Village (5°27′01.7″ latitude N, 10°05′27.5″ longitude E) located
in the town of Dschang, Menoua Division, in the West Region of Cameroon (Fig.1). Taxonomic identication
of the plant was made at the National Herbarium of Cameroon in comparison with a voucher specimen (A.J.M
Leeuwenberg No. 10196 registered at the National Herbarium under the No 48790/HNC). e popular use of
this plant as a biopesticide and as an insect repellent against pests in plantations and, as a repellent against A.
gambiae in the laboratory motivated the choice of this plant24,26.
T. diversifolia e essential oil was extracted in the inorganic chemistry
laboratory of the Faculty of Medicine and Pharmaceutical Sciences of the University of Douala, by hydrodistil-
lation using a Clevenger-type water steam apparatus.
Aer harvesting, fresh leaves (500g) of the plant were washed with distilled water then air-dried in the dark
for seven days at room temperature (29–33°C) with daily humidity of 15.54–27.93%.. e water content (Cwat
in %) was calculated using the following formula: Cwat = [((FVm−DPm)/FVm) × 100], where FVm is the fresh
vegetable mass (g) and DPm is the dry plant mass obtained (g).
Approximately 50kg of dried material were divided into 10 batches of 5kg and each batch was subjected to
distillation using a Clevenger-type water steam apparatus at 104°C for three hours aer the rst drop of distillate
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appeared. Once the distillation process is completed, the distillate was le to rest for 1h at room temperature
and aer this short stay in the separating funnel, two phases were formed. For each batch, 1mL of EO on top
of the water was collected with the micropipette and then introduced into a labelled bottle. e T. diversifolia
EO are collected in 10mL hermetically sealed glass vials wrapped with aluminum foil aer dehydration of the
oil-oral water mixture using anhydrous sodium sulfate (Na2SO4) then stored at + 4°C until further analysis.
e EO yield was calculated using the following formula: CEO = [(VEO/Md) × 100] ± [(VEO/Md) × 100], where
CEO is the EO yield (mL/g), VEO is the volume of EO collected (mL), VEO is the reading error, and Md the mass
of dried vegetal material (g).
T. diversifolia e composition
of volatile elements was determined using gas chromatography coupled with mass spectrometry (GC–MS). Sep-
aration of the EO components was performed on Hewlett-Packard silica fused capillary columns (50cm length,
0.32mm internal diameter, and 0.3µm lm thickness Carbowax). Helium was used as carrier gas at a ow rate
of 1mL/min. e GC column oven temperature varied from 60 to 240°C at a rate of 7°C/min for 20min. Mass
spectra were taken in scan mode in the range of 50–500m/z mass to charge, operated at 70eV, and the ion source
temperature was maintained at 250°C. e total run time was 23.82min. e identication of compounds was
done by comparing their retention index (RI) and mass spectra with those from the Wiley/National Bureau of
Standards, and the National Institute of Standards and Technology libraries stored in the GC–MS database. e
concentration of volatile compounds (Ci) was calculated using the following formula: %Ci = [(Ai × Fi)/Vol]
where, Ai is the peak air product, Fi is the proportionality factor and Vol is the volume injected.
A. coluzzii Eggs of A. coluzzii (Ngousso strain) were obtained from the “Organi-
sation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaounde”, Cameroon and reared
in plastic bowls (300 eggs per bowl) for 24h at the Insectarium of the Faculty of Medicine and Pharmaceutical
Sciences to obtain larvae. e dierent larval stages obtained were fed with sh food every morning. Once the
larval stages evolved into pupae, they were separated and kept in net-covered mosquito cages until emergence of
adults. A cotton piece soaked in a 10% glucose solution was placed inside the cages to feed the emerged adults.
e repellent potential of the T. diversifolia EO was evaluated by human landing assays
both in the eld against wild type mosquitoes and in the laboratory using established adult female A. coluzzii. All
volunteers freely received antimalarial prevention before experiments, as per the national guidelines for malaria
management27.
Figure1. Map showing (a) the geographical area of collection, (b) T. diversifolia in its natural habitat, and (c) T.
diversifolia essential oil-Photographs of the plants and its essential oil were provided by the authors.
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Repellency activity against wild‑type mosquitoes. Nightly captures of adult female mosquitoes were conducted
in April 2020 and among human volunteers in the Mabanda neighbor. Mabanda is a popular neighborhood
located in the third division of the city of Douala (Littoral region, Cameroon) and where insalubrity is very
present and the population lives in high promiscuity. It is also a neighborhood with a large number of mosquito
breeding sites due to human activities.
2mL of EO was dripped onto each leg from the bottom of the knee to the end of the ankle then was applied
by hand onto the legs of each volunteer, from the knee to the end of the ankle to cover a skin area of about 200
cm2 (0.01mL/cm2 of EO). e nine catchers were divided into three groups of three participants. e three
catchers from each group were placed one-meter away from each participant in the group while the distance
between groups was variable, depending on the capture sites (next to a stream between two plank houses, next
to the eld and next to the hard houses inland). In each group working simultaneously in one spot, the volunteer
received either C. citratus EO 100% known to have repellent properties (positive control), or T. diversifolia EO
100% (test group), or no substance (negative control) to control for any capture biases. C. citratus (citronella) is
greatly used as repellent by populations. In contrast, DEET repellents are few used by populations due to absence
of knowledge, cost and beliefs on its toxicity. In this context, we preferred using citronella-based repellents as
positive control for eld experiments. e capturers were instructed not to rub, touch, or wet their substance-
treated legs. e eld experiments were conducted between 7.00p.m. and 11.00p.m. (4-h exposure) with the
eld temperature varying from 29 to 30°C during each capture night. e volunteers had no contact with oils,
perfumed soaps, lotions or perfumes on the day of the assay. Mosquitoes species are counted and are identied
using morphological traits and dichotomous keys28–30.
Mosquito rearing and repellency activity against laboratory A. coluzzii strains. Larvae mosquitoes were fed daily
with 50mg sh powder for 6–7days. A piece of cotton wool soaked with glucose (30mL, 10%) was deposited
onto the net-covered for the nutrition of adults that emerged Cage. e repellency activity was assessed using the
Armin cage test as described by Schreck etal., and WHO guidelines on ecacy testing of mosquito repellent for
human skin31,32. Briey, 3–5-day-old and adult female A. coluzzii mosquitoes (n = 100) without blood supply for
one to two days were kept in a net-covered cage (35cm × 35cm × 35cm)24. e DEET 30% (positive control), or
vaseline (negative control), or one of the dierent concentrations of the T. diversifolia EO (10%, 30%, 50% and
100% in vaseline to reduce its volatility) were used to perform the assay as described previously24. Only the fore-
arm of each volunteer was exposed and the remaining area was protected with rubber gloves. e experiments
were conducted in two days between 8.00p.m. and 03.00 a.m., and were performed during a 1-h exposure period
in triplicate (three dierent human volunteers’ hands were used per test and each volunteer received the three
treatments at dierent times) with 1g of each formulation puting the EO treated arm and control arms into the
cages at the same time for a full hour. At the day 1, the positive control arm (DEET 30%) and the one treated with
30% EO were introduced simultaneously into the mosquito cage for each volunteer and at the day 2, the DEET
was replaced by the vaseline (negative control). e exposure was stopped when the rst bite of the volunteer was
noted. Before the experiment, the volunteer’s forearm was treated with petroleum jelly as control and exposed
for 30s to check for repulsion. If at least two mosquitoes landed on the arm, the repellency assay is continued as
this means that petroleum jelly has no repellent eect on mosquitoes, so it would not aect the repellent activity
of the EO of T. diversifolia to be tested. Likewise, the volunteers had no contact with other substances (i.e., body
oils and lotions, perfumed soaps, fragrances) on the day of the assay.
Repellency assay outcomes. Field and laboratory-based repellent activities were assessed by determining the
repulsion rate and the protection time for each substance. e repulsion rate (Re) was determined using the
following formula: Re = [(NC – NEO)/NC] × 100, where NC is the number of mosquitoes captured by volunteers
treated with negative control, and NEO is the number of mosquitoes captured by volunteers treated with the test
substance. is rate was used to assess reduction in the attractiveness of mosquitoes to humans treated with the
substance tested. e protection time is the time interval (min) between the application of the substance and the
rst mosquito bite.
Data were keyed in an Excel spreadsheet (Microso Oce, USA) and then exported to
the statistical package for social sciences v16 (SPSS, IBM, Inc., IL, Chicago, USA) and GraphPad v5.03 (Graph-
Pad Prism, Inc., San Diego, California, USA) soware for statistical analysis. Non parametric Mann–Whitney
and Kruskal–Wallis tests were used to compare mean values between groups while Fisher’s exact and Pearson’s
independence chi-square were used to compare proportions. Level of statistical signicance was set at p < 0.05.
e study was conducted in accordance with ethical
guidelines related to research on humans in Cameroon. e study received ethical clearance from the Insti-
tutional Committee of Ethics for Research for Human Health of the University of Douala (no. 1976/CEI-
UDo/06/2019/T). Before enrollment, subjects were informed on the purpose and process of the investigation
(background, goals, methodology, study constraints, data condentiality, and rights to opt out from the study),
and signed informed consent was obtained from all those who agreed to participate in the study in accordance
with the Helsinki Declaration. Participation was voluntary, anonymous and without compensation.
Collection of wild plant material was carried out in accordance with national guidelines and Cameroonian
legislation (provisions of the law on forest and environmental management in Cameroon No 94/01 of 20 Janu-
ary 1994 relating to the forest, fauna and shing regime in Cameroon and explicitly recognizing the rights of
local populations use on various forest products) and an identication certicate was obtained at the National
Herbarium of Cameroon.
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T. diversifolia e moisture content of the leaves of
T. diversifolia was 81% and the yellow essential oil yield was 0.02% aer hydrodistillation of 50kg of dry leaves
of T. diversifolia.
e GC–MS analysis revealed the presence of 29 compounds in the T. diversifolia EO (yellow pale-colored),
with -limonene (20.06%), α-Copaene (10.29%) and o-Cymene (10.0%) as the most represented (Table1). e
29 compounds identied belong to four groups namely monoterpenes, sesquiterpenes, phenylbutane deriva-
tives and triterpenes, which accounted for 54.13%, 35.22%, 8.87% and 1.78% of the compounds, respectively.
e mean time of protection against mosquito bites was signicantly higher in
volunteers treated with the C. citratus EO as compared to those treated with the T. diversifolia EO (125min
versus 71min, p = 0.04) (Fig.2).
e mean number of mosquitoes captured were similar between capturers treated with the C. citratus EO or
T. diversifolia EO (p = 0.60) (Fig.3a). Likewise, repulsion rate between the two groups were statistically similar
(92.1% for C. citratus–treated group, and 89.5% for T. diversifolia–treated group, p = 0.51) (Fig.3b).
e analysis of the entomofauna captured revealed a similar distribution of Culex, Anopheles and Mansonia
mosquitoes between the two groups treated with the EO (Fig.4). Regarding Anopheles mosquitoes collected in
the groups, these were predominantly identied as A. gambiae s.l.
It should be noted that minor burning sensations were reported only in volunteers treated with the C. citratus
EO. No adverse signs/symptoms were observed in those treated with the T. diversifolia EO.
Finding from the invitro repellency assay showed that the time of protec-
tion from biting was on average higher in the group of capturers treated with DEET as compared to the other
groups treated with varying concentration of the T. diversifolia EO, and the dierence was statistically signicant
Table 1. Chemical composition of the T. diversifolia essential oil. e table represents information on the
chemical composition of the T. diversifolia essential oil. RI: retention index; A number was used to dierentiate
between Sesquiterpernes (1 to 10), Monoterpenes (11 to 20), Phenylbutane derivatives (21 to 27) and the
Diterpenes (28 and 29). Percentage of each compound is presented in the last column.
No RI (min) C ompounds %
1 13.02 Bicyclo [7.2.0] undec-4-ene, 4,11,11-trimethyl-8-methylene-, [1R-(1R*, 4Z,9S*)] 0.68
2 13.16 Modephene 2.28
3 13.28 (1R, 3aS,5aS,8aR)-1,3a,4,5a-Tetramethyl-1,2,3,3a,5a,6,7,8-octahydrocyclopenta[c]pentalene 0.30
4 13.63 α-Copaene 10.29
5 13.87 Caryophyllene 8.44
6 15.63 (E)-2-((8R,8aS)-8-8aDimethyl-3,4,6,7,8,8a-hexahydronaphtalen-2-1H)-ylidene) propan-1-ol 1.07
7 16.22 Isocaryophilene 8.73
8 16.64 1a,2,6,7,7a,7b-hexahydro-1,1,7,7a-tetramethyl-, [1aE-(1aà,7à, 7aà,7ba]-1H-cycloprapa[a]naphthalene 1.58
9 16.76 o-Cymene 10.00
10 17.89 α-Guaiene 2.14
11 4.67 α-Pinene 7.72
12 6.29 -Limonen 20.06
13 6.82 (+)-3-Caren 0.91
14 7.56 Naphtalen 4.01
15 8.53 1,3,8-p-Menthetriene 0.85
16 9.42 α-Terpineol 1.86
17 10.03 2,6,6-trimethyl-1-Cyclohexane-1-carboxaldehyde 0.86
18 10.90 Cis-p-Mentha-2,8-dien-1-ol 0.91
19 14.47 Oxide-1-aromadendrene 1.90
20 14.93 n-Pentadecanol 8.77
21 5.45 6-Methyl-5-Hepten-2-one 2.76
22 7.63 Nonanal 0.31
23 8.71 2,6-Dimethyl-3,5-Heptadien-2-ol 0.83
24 9.17 4-Terpinenyl acetate 0.10
25 14.11 4-(2,6,6-Trimethyl-1-cyclohewen-1-yl)-2-Butanone 0.61
26 20.57 6,10,14-Trimethyl-2-Pentadecanone 4.26
27 25.30 2-Methyl-eicosane 0.86
28 21.67 3-Methyl-2-(3,7,11-trimethyldodecyl) furane 0.88
29 24.25 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 0.90
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(p = 0.0016) (Fig.5a). In contrast, no dierence was found in terms of the number of mosquitoes captured and
repulsion rate between DEET-treated group and the T. diversifolia–treated groups (p > 0.05) (Fig.5b,c). No aller-
gic events were reported during the laboratory experiments.
Figure2. Mean protection time on eld of the dierent substances tested. EO(Cc): Essential oil of C. citratus
(positive control); EO(Td): Essential oil of T. diversifolia; Mann–Whitney test was used to make pairwise
comparisons; *Statistically signicant at p < 0.05.
Figure3. Mean number of mosquitoes captured (a), and mean repulsion rate (b) in the dierent groups
evaluated. EO (Cc): Essential oil of C. citratus (positive control); EO (Td): Essential oil of T. diversifolia; Pe arson’s
independence chi-square test was used to compare the groups; *statistically signicant at p < 0.05.
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e emergence of mosquito populations resistant to synthetic insecticides and commercially available repel-
lents hinders their use at the population level for control of mosquito-borne diseases such malaria. Unlike their
synthetic counterparts, there is no evidence on emergence of resistance to natural substances. In this context,
there is a need for new molecules with good repellent potentials and safety. We therefore evaluated the repellent
potential of EO from leave parts of T. diversifolia, a plant traditionally used in Cameroon for treating chickenpox,
and in other countries (e.g., Mexico and Nigeria) for treating malaria and other diseases32–35.
e yield of T. diversifolia EO (0.02%) diers from that found for the plant collected in another city in Cam-
eroon in a previous study36. is nding is consistent with other previous studies showing a dierence in yields
depending on plant’s harvest location, harvest period, leaf condition, drying or extraction time or technique
with a yield that varies between 0.01% and 0.1% for essential oils extracted from leaves24,37–39. Another study
revealed that in addition to the storage time, the transport conditions also impact the EO yield of this plant40.
Hydrogenated monoterpenes and sesquiterpenes were the main compounds found in the T. diversifolia EO.
is nding is also consistent with previous reports on the chemical composition of the plant EO from other
regions of Cameroon and beyond24,33,35,38,41, though other previous reports from Cameroon outlined a predomi-
nance of sesquiterpenes in ower EO36. Our detailed analysis of the chemical composition identied 3 of the
29 compounds at high levels viz. ()-α-limonene (monoterpenes), α-Copaene (sesquiterpenes) and o-Cymene
(sesquiterpenes). Lamaty and colleagues found that (Z)-β-ocimene (40.2%) was the main compound from EO
of T. diversifolia collected in the town of Yaoundé, Centre region of Cameroon36. is discrepancy highlights the
role of environment in shaping biological development of plants, and thus its impact on their chemical composi-
tion and biological activities24,37,41.
Our study is the rst to evaluate the repellent activity of T. diversifolia against A. coluzzii, a major malaria
vector in Africa. We demonstrated the repellent potential of this plant against laboratory strains of A. coluzzii
and against natural mosquito bites in eld studies. Our ndings support previous studies on the same plant that
reported repellent activities of its EO fractions against A. gambiae, Aedes aegypti and Culex quinquefasciatus23.
is repellent activity in natural condition was higher than that observed against the laboratory strain, probably
due to the time between the harvest, the time of extraction (4months aer harvest) of essential oil, the natural
condition test (15days aer extraction) and the laboratory test (1month aer the natural conditions test) that
would have an impact on the volatile properties of certain terpenic compounds38. e high content of C. citratus
EO in α-pinene (34.4% vs 7.72% for T. diversifolia EO) could explain it strong repellent activity (92% vs 89.5%)
compared to the T. diversifolia EO rich in ()-α-limonene, and studies have shown that α-pinene is a powerful
repellent42–44. e dierence in the duration between the harvest period and the extraction could have induced
the volatility of compounds (dierence in concentration) and the conformation change of certain compounds
of the T. diversifolia EO tested in our study, as has been suggested by Walker etal.41 e latter could also explain
the low repellent activity of T. diversifolia EO compared to C. citratus EO. Besides, it would also be interesting to
conduct further studies to identify the chemical compounds responsible for repellent activity of T. diversifolia
along with elucidating mechanism of action. Given high content of ()-α-limonene, this compound is probably
greatly involved in the repellent activity of T. diversifolia EO, possibly in association with other major compounds
found (α-Copaene and o-Cymene).
e results showed a shorter protection time in T. diversifolia EO-treated volunteers compared to the individu-
als treated with the positive control for both the laboratory (DEET) and eld (C. citratus EO) assays. In natural
conditions, the mean protection time was 71min for T. diversifolia EO and 125min for the positive control. ese
ndings suggest that the T. diversifolia EO has similar repellency, but that the volatile nature of its compounds
greatly impacted the protection time of T. diversifolia EO. ese preliminary results on protection time against
wild mosquitoes suggest that T. diversifolia EO should be used each hour in to maintain its repellent activity,
Figure4. Entomofauna captured in the dierent groups evaluated. EO(Cc): Essential oil of C. citratus (positive
control); EO(Td): Essential oil of T. diversifolia; Pearson’s independence chi-square test was used to compare the
groups; *statistically signicant at p < 0.05.
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Figure5. Findings from the invitro repellency activity. (a) Mean protection time, (b) Number of mosquitoes
captured aer 1h, (c) Repulsion rate aer 1h. DEET: N, N-diethyl-3-methylbenzamide (positive control); EO
(Td-X%): Essential oil of T. diversifolia at dose 10%, 30%, 50% and 100%; Kruskal–Wallis test was used to make
comparisons between the groups; *Statistically signicant at p < 0.05.
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which is not achievable in practice. In this context, it would be helpful to develop release-control formulations
in order to reduce its the volatility.
In the laboratory, the concentrations of the T. diversifolia EO at 50% provided a better time protection
(11.9min) against A. coluzzii than the 100% extract (9.6min), but this time was signicantly less than that of
DEET at 30%. Oyewole etal. in Nigeria24 had found protection times of 120, 160 and 210min, respectively for the
10%, 50% and 100% formulations, higher than those found in our study. ese results corroborate with previous
study suggesting the inuence of the solvent used in the formulations; unlike hexane which was used by Oyewole
etal., the petroleum jelly used in our study would slow the volatility of the EO24. Variability in the sensitivity
of an EO against Anopheles species has also been reported, A. gambiae was more sensitive to repellents than A.
coluzzii18. One solution to improve protection time of T. diversifolia EO could be to develop controlled-release
formulations in order to increase the duration of repellence activity. e volatile nature of the compounds of the
EO as well as a combination of other factors such as chemical composition, natural resistance of the mosquito
vector species and experimental conditions can explain discrepancies observed between the groups treated and
positive control.
e present preliminary study showed promising repellent activity of the leave T. diversifolia EO against A.
coluzzii and, suggests that it could be used as an eective vector control tool at the individual level to comple-
ment conventional control methods at the community level. However, the limited time of protection compared
to controls outlines the need for extensive research on development of release-control formulations and inocuity
before its potential introduction as a commercial repellent.
All data underlying the ndings and the conclusions are included within the manuscript.
Received: 29 September 2021; Accepted: 17 March 2023
1. Coetzee, M. et al. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa 3619,
246–274 (2013).
2. Cowman, A. F., Healer, J., Marapana, D. & Marsh, K. Malaria: Biology and disease. Cell 167(3), 610–624. https:// doi. or g/ 10. 1016/j.
cell. 2016. 07. 055 (2016).
3. World Health Organization (WHO). World Malaria Report 2020: 20 years of global progress & challenges le:///C:/Users/
DREBOU~1/AppData/Local/Temp/9789240015791-eng.pdf (WHO, 2020).
4. World Health Organization (WHO). Artemisinin resistance and artemisinin-based combination therapy ecacy: status report
https:// apps. who. int/ iris/ handle/ 10665/ 274362 (WHO, 2018).
5. Antonio-Nkondjio, C. et al. Review of the evolution of insecticide resistance in main malaria vectors in Cameroon from 1990 to
2017. Parasit. Vectors. 10(1), 472. https:// doi. org/ 10. 1186/ s13071- 017- 2417-9 (2017).
6. Moiroux, N. et al. Changes in Anopheles funestus biting behavior following universal coverage of long-lasting insecticidal nets in
Benin. J. Infect. Dis. 206(10), 1622–1629. https:// doi. org/ 10. 1093/ infdis/ jis565 (2012) (Epub 2012 Sep 10).
7. Reddy, M. R. et al. Outdoor host seeking behaviour of Anopheles gambiae mosquitoes following initiation of malaria vector control
on Bioko Island, Equatorial Guinea. Malar J. 10, 184. https:// doi. org/ 10. 1186/ 1475- 2875- 10- 184 (2011).
8. Russell, T. L. et al. Increased proportions of outdoor feeding among residual malaria vector populations following increased use
of insecticide-treated nets in rural Tanzania. Malar J. 10, 80. https:// doi. org/ 10. 1186/ 1475- 2875- 10- 80 (2011).
9. Cameroon National Public Health, Observatory and World Health Organization. Monitoring report of 100 key health indicators
in Cameroon in 2019 & SDG focus. French le. Accessed in 2019. ht tps:// www. minsa nte. cm/ site/?q= fr/ conte nt/ rappo rt- de- suivi-
des- 100- indic ateurs- cl% C3% A9s- de- sant% C3% A9- au- camer oun- en- 2019- focus- odd (2019).
10. World Health Organization and Rollback malaria Partenership. High burden to high impact: A targeted malaria response http://
apps. who. int/ iris/ bitst ream/ handle/ 10665/ 275868/ WHO- CDS- GMP- 2018. 25- eng. pdf? ua=1 (WHO, 2018, revised March 2019).
11. MbohouNchetnkou, C., KojomFoko, L. P. & Lehman, L. G. Knowledge, attitude, and practices towards malaria among employees
from enterprises in the town of Douala, Cameroon. Biomed. Res. Int. 2020, 8652084. https:// doi. org/ 10. 1155/ 2020/ 86520 84 (2020).
12. Cameroon Health Ministry, National Institute of Statistics. Post-campaign survey on the use of long-acting insecticide-treated
mosquito nets. French le. Accessed in February 2019. http:// www. sante tropi cale. com/ rappo rts. asp? action= lire& id= 773& speci
alite= Palud isme (2013).
13. Cameroon Health Ministry. Plan National de développement sanitaire (PNDS) 2011–2015. 152. French le. Accessed in February
2021. https:// extra net. who. int/ nutri tion/ gina/ sites/ defau lt/ les store/ 2011% 20CMR% 20PNDS. pdf (2011).
14. Maia, M. F. & Moore, S. J. Plant-based insect repellents: A review of their ecacy, development and testing. Malar J. 10(Suppl 1),
S11. https:// doi. org/ 10. 1186/ 1475- 2875- 10- S1- S11. PMID: 21411 012; PMCID: PMC30 59459 (2011).
15. Qiu, H., Jun, H. W. & McCall, J. W. Pharmacokinetics, formulation, and safety of insect repellent N,N-diethyl-3-methylbenzamide
(deet): A review. J. Am. Mosq. Control Assoc. 14(1), 12–27 (1998).
16. Abiy, E., Gebre-Michael, T., Balkew, M. & Medhin, G. Repellent ecacy of DEET, MyggA, neem (Azedirachta indica) oil and
chinaberry (Melia azedarach) oil against Anopheles arabiensis, the principal malaria vector in Ethiopia. Malar J. 14, 187. https://
doi. org/ 10. 1186/ s12936- 015- 0705-4 (2015).
17. Yoon, J. K. et al. Comparison of repellency eect of mosquito repellents for DEET, Citronella, and Fennel Oil. J. Parasitol. Res.
2015, 361021. https:// doi. org/ 10. 1155/ 2015/ 361021 (2015) (Epub 2015 Oct 7).
18. Adio, N. A. et al. Eectiveness of organic insecticides based on essential oil of Cymbopogon schoenanthus (l.) spreng against red
bugs (Dysdercus voelkeri, schmidt) in cotton culture in Togo. AJFAND. 21(3), 17727–17740. https:// doi. org/ 10. 18697/ ajfand. 98.
20095 (2021).
19. Oshaghi, M. A. et al. Repellent eect of extracts and essential oils of Citrus limon (Rutaceae) and Melissa ocinalis (Labiatae)
against main malaria vector, Anopheles stephensi (Diptera: Culicidae). Iran J. Public Health. 32(4), 47–52 (2003).
20. World Health Organization (WHO). Malaria Entomology and Vector Control: A Participant’s Guide. French le. Accessed in
February 2021. le:///C:/Users/DREBOU~1/AppData/Local/Temp/9789242505818_fre.pdf (WHO, 2014).
21. Maina, G. J., Timothy, M. & Muchunu, M. J. Antieas activity and safety of Tithonia diversifolia and Senna didymobotrya extracts.
J. Pharm. Pharmacol Res. 2(3), 078–092. https:// doi. org/ 10. 26502/ jppr. 0012 (2018).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
10
Vol:.(1234567890)
| (2023) 13:6001 | https://doi.org/10.1038/s41598-023-31791-6
www.nature.com/scientificreports/
22. Asadollahi, A., Khoobdel, M., Zahraei-Ramazani, A., Azarmi, S. & Mosawi, S. H. Eectiveness of plant-based repellents against
dierent Anopheles species: A systematic review. Malar J. 18(1), 436. https:// doi. org/ 10. 1186/ s12936- 019- 3064-8 (2019).
23. Obiakara, M. C. & Fourcade, Y. Climatic niche and potential distribution of Tithonia diversifolia (Hemsl.) A. Gray in Africa. PLoS
ONE 13(9), e0202421. https:// doi. org/ 10. 1371/ journ al. pone. 02024 21 (2018).
24. Oyewole, I. O. et al. Anti-malarial and repellent activities of Tithonia diversifolia (Hemsl.) leaf extracts. J. Med. Plant Res. 2(8),
171–175 (2008).
25. Tchoumbougnang, F. et al. Larvicidal activity against Anopheles gambiae Giles and chemical composition of essential oils from
four plants cultivated in Cameroon. Biotechnol. Agron. Soc. Environ. 13(1), 77–84 (2009).
26. Wanzala W, Mukabana R., Hassanali A. e eect of essential oils of Tagetes minuta and Tithonia diversifolia on on-host behaviour
of the brown ear tick Rhipicephalus appendiculatus. Livestock Res. Rural Dev. 30(6) (2018).
27. Cameroon National Public Health. Guide to the management of malaria in Cameroon for the use of health personnel. Yaoundé.
Accessed in March 2020. French le. http:// cdnss. minsa nte. cm/ sites/ defau lt/ les/ GUIDE% 20PEC% 20% 20PAL UDISME% 202019%
2018% 20JUIN. pdf (2019).
28. Rueda, L. M. Pictorial Keys for the Identication of Mosquitoes (Diptera: Culicidae) Associated with Dengue Virus Transmission
(Magnolia Press, 2004) ISBN 1-877354-47-3.
29. Gillies, M. T. & Coetzee, M. A supplement to the Anophelinae of Africa south of the Sahara (Ethiopian zoogeographical region).
South Afr. Inst. Med. Res. 55, 1–146 (1987).
30. Gillies, M. & De Meillon, B. e Anophelinae of Africa South of the Sahara (Ethiopian Zoogeographical Region) 2nd edn. (South
Afr Inst Med Res, 1968).
31. Schreck, C. E. Techniques for the evaluation of insect repellents: A critical review. Annu. Rev. Entomol. 22, 101–119. https:// doi.
org/ 10. 1146/ annur ev. en. 22. 010177. 000533 (1977).
32. World Health Organization (WHO). Guidelines for ecacy testing of mosquito repellents for human skins https:// apps. who. int/
iris/ handle/ 10665/ 70072 and https:// apps. who. int/ iris/ bitst ream/ handle/ 10665/ 70072/ WHO_ HTM_ NTD_ WHOPES_ 2009.4_
eng. pdf? seque nce= 1& isAll owed=y (WHO, 2009).
33. Mabou Tagne, A., Marino, F. & Cosentino, M. Tithonia diversifolia (Hemsl.) A. Gray as a medicinal plant: A comprehensive review
of its ethnopharmacology, phytochemistry, pharmacotoxicology and clinical relevance. J. Ethnopharmacol. 220, 94–116. https://
doi. org/ 10. 1016/j. jep. 2018. 03. 025 (2018).
34. World Health Organization (WHO). Procedures for testing insecticide resistance in mosquito vectors of malaria. 2nd ed. French
le. https:// apps. who. int/ iris/ bitst ream/ handle/ 10665/ 259741/ 97892 42511 574- fre. pdf (WHO, 2017).
35. Ajao, A. A. & Moteetee, A. N. Tithonia diversifolia (Hemsl) A. Gray. (Asteraceae: Heliantheae), an invasive plant of signicant
ethnopharmacological importance: A review. South Afr. J. Bot. 113, 396–403. https:// doi. org/ 10. 1016/j. sajb. 2017. 09. 017 (2017).
36. Lamaty, G. et al. Aromatic plants of tropical central Africa. III. Constituents of the essential oil of the leaves of Tithonia diversifolia
(Hemsl.) A. Gray from Cameroon. J. Essent. Oil Res. 3(6), 399–402. https:// doi. org/ 10. 1080/ 10412 905. 1991. 96979 73 (1991).
37. Ogundare, A. O. Antimicrobial eect of Tithonia diversifolia and Jatropha gossypifolia leaf extracts. Trends Appl. Sci. Res. 2, 145–150.
https:// doi. org/ 10. 3923/ tasr. 2007. 145. 150 (2007).
38. Gu, J. Q. et al. Sesquiterpenoids from Tithonia diversifolia with potential cancer chemopreventive activity. J. Nat. Prod. 65(4),
532–536. https:// doi. org/ 10. 1021/ np010 545m (2002).
39. Moronkola, D. O., Ogunwande, I., Walker, T. M., Setzer, W. N. & Oyewole, I. A. Identication of the main volatile compounds
in the leaf and ower of Tithonia diversifolia (Hemsl) Gray. J. Nat. Med. 611, 63–66. https:// doi. org/ 10. 1007/ s11418- 006- 0019-5
(2006).
40. Soro, L. C. et al. Variability of the chemical composition of the essential oil of Lippia multiora leaves cultivated in Ivory Coast. J.
Appl. Bio. Syst. 88, 8180–8193 (2015).
41. Aa, B. F., Wognin, E. & Tonzibo, Z. F. Chemical variability of Tithonia diversifolia (hemsl.) A. Gray leaf and stem oil from Côte
d’ivoire. Int. J. Pharm. Sci. Res. 6(5), 2214–2222. https:// doi. org/ 10. 13040/ IJPSR. 0975- 8232. 6(5). 2214- 22 (2015).
42. Aizpurua-Olaizola, O. et al. Evolution of the Cannabinoid and Terpene content during the growth of cannabis sativa plants from
dierent chemotypes. J. Nat. Prod. 79(2), 324–331. https:// doi. org/ 10. 1021/ acs. jnatp rod. 5b009 49 (2016) (Epub 2016 Feb 2).
43. Bokobana, E. M. et al. Evaluation of the insecticidal and repellent potential of the essential oil of Cymbopogon schoenanthus Spreng
on Aphis gossypii glover (Homoptera: Aphididae), a pest of cotton in Togo. REV. CAMES, 2(2) (2014). ISSN: 2424-7235. French
le. http:// publi cation. lecam es. org/ index. php/ svt/ artic le/ viewF ile/ 421/ 280 (2021).
44. Menut, C., Lamaty, G., Zollo, P. H. A., Kuiate, J. R. & Bessière, J. M. Aromatic plants of tropical central Africa. IX. Chemical com-
position of ower essential oils of Tithonia diversifolia (Hemsl.) A. Gray from Cameroon. J. Essent. Oil Res. 6(4), 651–653. https://
doi. org/ 10. 1080/ 10412 905. 1992. 96981 53 (1992).
We are very grateful to the participants who agreed to participate in this study as well as the technical sta of the
Inorganic Chemistry and Parasitology Labs of the Faculty of Medicine and Pharmaceutical Sciences for their
support and cooperation during the survey.
Conceptualization: C.A.T., C.N., C.E.E.M. Data curation: C.A.T., L.P.K.F., E.E.S. Formal analysis: L.P.K.F.,
C.E.E.M. Investigation: C.A.T., C.N. Methodology: C.A.T., C.N., C.E.E.M. Resources: G.T., E.E.S. Supervision:
C.N., C.E.E.M. Validation: C.N., C.E.E.M. Visualization: C.N., F.E.M., C.E.E.M. Writing—original dra: C.A.T.,
L.P.K.F., C.E.E.M. Writing—review and editing: C.A.T., C.N., L.P.K.F., C.N., E.E.S., G.T., F.E.M., A.L., C.E.E.M.
Consent to publish has been obtained from all included persons in the study.
e authors declare no competing interests.
Correspondence and requests for materials should be addressed to C.E.E.M.
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