insects

Article
Study of the Repellent Activity of 60 Essential Oils and Their
Main Constituents against Aedes albopictus, and
Nano-Formulation Development
Weifeng Wu 1 , Yu Yang 1 , Yingmiao Feng 2,3 , Xiaofei Ren 1 , Yuling Li 1 , Wenjiao Li 1 , Jietong Huang 1 ,
Lingjia Kong 1 , Xiaole Chen 1,4,5 , Zhongze Lin 3 , Xiaohui Hou 6 , Longlai Zhang 7 , Yajie Chen 1 ,
Zhaojun Sheng 1,5, * and Weiqian Hong 1,5,8, *

1 School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China
2 The Third Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, China
3 Faculty of Southern Medicine, Guangdong Jiangmen Chinese Medicine College, Jiangmen 529000, China
4 Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China
5 International Healthcare Innovation Institute (Jiangmen), Jiangmen 529020, China
6 School of Preclinical Medicine, Zunyi Medical University, Zunyi 563003, China
7 MHOME (Guangzhou) Industrial Co., Ltd., Guangzhou 510700, China
8 Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, UK
* Correspondence: wyuchemszj@126.com (Z.S.); davidhwq@liverpool.ac.uk (W.H.)

Simple Summary: Due to the environment and human health concerns of synthetic repellents,
essential oils (EOs) as natural alternatives have received increased attention. In this study, the
repellent activity of 60 commercial EOs against Aedes albopictus was evaluated. In the initial screening,
8 active EOs including cinnamon, marjoram, lemongrass, bay, chamomile, jasmine, peppermint2, and
thyme were selected. Twenty-one major constituents (>5% relative area) in the 8 active EOs were
identified via gas chromatography-mass spectrometry (GC-MS) analysis. Cinnamaldehyde, citral
Citation: Wu, W.; Yang, Y.; Feng, Y.; and terpinene-4-ol displayed the highest repellent activity with more than 60% RR, which were more
Ren, X.; Li, Y.; Li, W.; Huang, J.; Kong, active than N,N-diethyl-3-methyl benzoyl amide (DEET). Next, their nanoemulsions were prepared
L.; Chen, X.; Lin, Z.; et al. Study of and characterized. In the arm-in-cage assay, cinnamaldehyde- and citral-based nanoemulsions have
the Repellent Activity of 60 Essential
prolonged mosquito protection time compared with their normal solutions.
Oils and Their Main Constituents
against Aedes albopictus, and
Abstract: Mosquitoes are one of the most important disease vectors from a medical viewpoint in that
Nano-Formulation Development.
they transmit several diseases such as malaria, filariasis, yellow and Dengue fever. Mosquito vector
Insects 2022, 13, 1077. https://
control and personal protection from mosquito bites are currently the most efficient ways to prevent
doi.org/ 10.3390/insects13121077
these diseases. Several synthetic repellents such as DEET, ethyl butylacetylaminopropionate (IR3535)
Academic Editor: Lukasz L. Stelinski and 1-(1-methylpropoxycarbonyl)-2-(2-hydroxyethyl)piperidine) (Picaridin), have been widely used
Received: 12 October 2022 to prevent humans from receiving mosquito bites. However, the use of synthetic repellents has
Accepted: 17 November 2022 raised several environment and health concerns. Therefore, essential oils (EOs) as natural alternatives
Published: 22 November 2022 receive our attention. In order to discover highly effective mosquito repellents from natural sources,
the repellent activity of 60 commercial EOs against Ae. albopictus was screened in this study. Eight EOs
Publisher’s Note: MDPI stays neutral
including cinnamon, marjoram, lemongrass, bay, chamomile, jasmine, peppermint2, and thyme,
with regard to jurisdictional claims in
published maps and institutional affil-
showed a suitable repellent rate (>40%) at the tested dose of 10 µg/cm2 . Then, their main constituents
iations. were analyzed by GC-MS, and the active constituents were identified. The most active compounds
including cinnamaldehyde, citral and terpinen-4-ol, exhibited an 82%, 65% and 60% repellent rate,
respectively. Moreover, the nanoemulsions of the three active compounds were prepared and
characterized. In the arm-in-cage assay, the protection times of the nanoemulsions of cinnamaldehyde
Copyright: © 2022 by the authors. and citral were significantly extended compared with their normal solutions. This study provides
Licensee MDPI, Basel, Switzerland.
several lead compounds to develop new mosquito repellents, and it suggests that nanoemulsification
This article is an open access article
is an effective method for improving the duration of the activity of natural repellents.
distributed under the terms and
conditions of the Creative Commons
Keywords: essential oil; repellent; Aedes albopictus; nanoemulsion
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Insects 2022, 13, 1077. https://doi.org/10.3390/insects13121077 https://www.mdpi.com/journal/insects

Insects 2022, 13, 1077 2 of 13

1. Introduction
Mosquito bites not only cause several allergic reactions including itching and swelling,
but are also a mechanism to transmit pathogens between people and animals [1].
Ae. albopictus (Skuse), also known as Asian tiger mosquito, is a key carrier of dengue,
Zika and yellow fever viruses [2]. Mosquito control and personal protection from mosquito
bites are currently the most important measures to prevent these diseases [3]. One of the
most common approaches is to use insect repellents. Insect repellents can be classified into
two categories: spatial and contact repellents. Their action modes are different. Spatial
repellents, such as some synthetic pyrethroids and botanical compounds, are generally
highly volatile and capable of diffusing through the air in treated regions [4]. The repellent
vapors from spatial repellents lead to the aversive behavior and deleterious physiological
response of host-seeking mosquitoes [5,6]. Contact repellents such as DEET, picaridin, and
IR3535, are capable of attenuating the antennal responses of mosquitoes to various human
and veterinary attractive odorants via direct inhibition or attenuation of action potential
amplitudes or frequencies emanating from olfactory receptor neurons [6]. Nowadays, the
most used synthetic repellents represented by DEET are contact repellents, which need to
be applied directly on the human skin. The use of synthetic repellents to control mosquitoes
raises several concerns related to the environment and human health [7]. On the contrary,
natural essential oils (EOs) have advantages such as the wide spectrum of efficacy against
mosquitoes, multiple mode of actions, low residue and low toxicity [8,9], thereby they have
received our attention.
EOs are defined as volatile oils that have strong aromatic components and give a
distinctive odor, flavor or scent to an aromatic plant [10]. Many natural EOs with the
function of repelling mosquitoes have been discovered and exploited [7,11]. Among
them, citronella oil was the most widely used before the 1940s, and is still used today in
many formulations [12]. Others include eucalyptus, clove, lavender and lemon oils [3,13].
In modern times, the development of natural repellents was neglected after the appearance
and rapid development of synthetic repellents. However, with increasing attention and
demand for healthy and environmentally friendly mosquito repellents, the research and
development of natural EOs as mosquito repellents have begun to speed up again [14].
In our previous study, the larvicidal activity and synergistic effect with deltamethrin of
53 commercial EOs were screened and studied [15]. In order to discover highly effec-
tive mosquito repellents from natural sources and to investigate the further applications
of EOs in mosquito-vector control, the repellent activity of 60 commercial EOs against
Ae. albopictus was evaluated in this study. The active constituents of the active EOs
were identified. Moreover, the nanoformulation was used to improve the duration of
natural repellents.

2. Materials and Methods

2.1. Essential Oils and Reagents
The information of 60 EOs is listed in Table S1 of supporting information. Lemon
eucalyptus EO was purchased from Jiangxi Hualong Plant Flavor Co., Ltd., Ji’an, China;
and other EOs were purchased from Guangzhou Yuxitang Cosmetics Co., Ltd. Guangzhou,
China. All were pure EOs with no additive.
Benzyl acetate (purity: 95%), 2,2,4,6,6-pentamethyl-heptane (98%), menthol (97%),
terpinen-4-ol (95%), cinnamaldehyde (98%), citral (isomers of neral and geranial, 98%),
p-menthone (cis-&trans-isomers, 98%) and diethyl phthalate (99%) were purchased from
Bidepharm (Shanghai, China). p-Cymene (97%) was purchased from Energy Chemical
(Shanghai, China). Limonene (95%), linalool (96%), thymol (99%), carvacrol (98%), eugenol
(99%), and β-caryophyllene (90%) were purchased from TCI (Shanghai, China). γ-Terpinene
(95%), PEG-40 (HLB value: 13-14; pH value: 5.0-7.0), 1,2-propanediol (99%) were pur-
chased from Macklin (Shanghai, China). α-Terpineol (96%) was purchased from Alfa Aesar
(UK). (E)-2-Hexyl- cinnamaldehyde (92%) and DEET (99%) were purchase from Aladdin
 Bidepharm (Shanghai, China). p-Cymene (97%) was purchased from Energy Chemical
(Shanghai, China). Limonene (95%), linalool (96%), thymol (99%), carvacrol (98%),
eugenol (99%), and β-caryophyllene (90%) were purchased from TCI (Shanghai, China).
γ-Terpinene (95%), PEG-40 (HLB value: 13-14; pH value: 5.0-7.0), 1,2-propanediol (99%)
were purchased from Macklin (Shanghai, China). α-Terpineol (96%) was purchased from
Insects 2022, 13, 1077 Alfa Aesar (UK). (E)-2-Hexyl- cinnamaldehyde (92%) and DEET (99%) were purchase 3 of 13
from Aladdin (Shanghai, China). Diisobutyl phthalate (99%) was purchased from J&K
Scientific (Beijing, China). Defibrinated sheep blood was purchased from Rigorous
Scientific (Guangzhou,
(Shanghai, China).
China). Diisobutyl phthalate (99%) was purchased from J&K Scientific (Beijing, China).
Defibrinated sheep blood was purchased from Rigorous Scientific (Guangzhou, China).
2.2. Mosquitoes
2.2. Mosquitoes
The Ae. albopictus larvae were collected from Jiuwei Village, Huangpu District of
The Ae.ofalbopictus
Guangzhou larvae were
China (latitude: collected
23°06′24.17″ N, from Jiuwei113°26′43.07″
longitude: Village, Huangpu District
E) in March of
2013.
Guangzhou of China (latitude: 23 ◦ 060 24.1700 N, longitude: 113◦ 260 43.0700 E) in March 2013.
The adult mosquitoes that emerged from these larvae were identified as Ae. Albopictus
The adult mosquitoes
according that emerged
to the morphology. The from these larvae
mosquitoes werewere identified
reared in the as Ae. Albopictus
insectary of the
according
Internationalto the morphology.
Healthcare The mosquitoes
Innovation were reared
Institute (Jiangmen) in theThe
of China. insectary of the
insectary wasInter-
kept
national Healthcare Innovation Institute (Jiangmen) of China. The insectary
at 26 ± 2 °C, 70 ± 5% relative humidity with a photoperiod of 14 h light and 10 h dark. was kept at
26 ± 2 ◦ C, 70 ± 5% relative humidity with a photoperiod of 14 h light and 10 h dark. Larvae
Larvae were fed daily with fish food, and adults were fed with 5% glucose solution. 5 to
were fed daily
7-day-old with
female fish food, and
mosquitoes wereadults
used were
in thefed with 5% glucose solution. 5 to 7-day-old
bioassays.
female mosquitoes were used in the bioassays.
2.3. Repellent Activity Bioassay
2.3. Repellent Activity Bioassay
The repellent activity of 60 EOs was tested using the Hemotek membrane feeding
The repellent activity of 60 EOs was tested using the Hemotek membrane feeding
system (Discovery Workshops, Accrington, UK), and following the reported method
system (Discovery Workshops, Accrington, UK), and following the reported method [16,17],
[16,17], with some modification. As shown in Figure 1, 2.5 mL of sterile defibrinated sheep
with some modification. As shown in Figure 1, 2.5 mL of sterile defibrinated sheep blood
blood was placed in the feeding room. The blood was preheated and kept at 37.5 °C to
was placed in the feeding room. The blood was preheated and kept at 37.5 ◦ C to simulate
simulate the blood temperature of human beings. Parafilm was used to seal the blood and
the blood temperature of human beings. Parafilm was2 used to seal the blood and simulate
simulate the skin. The exposed surface was 15.9 cm . 159 μg repellent was dissolved in
the skin. The exposed surface was 15.9 cm2 . 159 µg repellent was dissolved in acetone,
acetone, giving the testing solution. The solution was uniformly loaded on 15.9 cm2 of
giving the testing solution. The solution was uniformly loaded on 15.9 cm2 of cotton net,
cotton
and net,
then theand
netthen the netatwas
was dried room dried at room for
temperature temperature for 10 minthe
10 min to evaporate to acetone.
evaporate the
Next,
acetone. Next, the net was fixed and covered on the parafilm. The same cotton
the net was fixed and covered on the parafilm. The same cotton net loaded with pure net loaded
with pure
acetone wasacetone
used inwas
theused in the
control control group.
group.

Figure 1. Repellent activity test device.

When the bioassay began, 20 to 30 female mosquitoes were put in a disposable cup
without a bottom. The cup was sealed with a movable plastic baffle, which controlled the
exposure status of mosquitoes to repellent and blood. Female mosquitoes were exposed for
30 min. After that, the test mosquitoes were immobilized using CO2 , and transferred to
the fridge to freeze. Then, the numbers of blood-fed and unfed mosquitoes were recorded.
The repelling rate (RR) was calculated using Equation (1) [17]. Three replicates were carried
out for each sample, and the average RR was calculated.

F0 /T0 − F/T
RR (%) = × 100% (1)
F0 /T0
Insects 2022, 13, 1077 4 of 13

where F0 and T0 are the numbers of fed mosquitoes and all mosquitoes in the control group,
respectively. F and T are those in the test group.

2.4. Chemical Compositions Analysis

The chemical compositions of EOs were analyzed using GC-MS (Thermo Scientific,
TRACE 1300 GC coupled to an ISQ Qd Mass Spectrometer and equipped with a TG-5 MS
capillary column). The analysis method used follows that of Sheng et al. [15]. Based on the
search results of National Institute of Standards and Technology (NIST) mass spectral data
library, the constituents were identified through Kovats retention indices (RI) combined
with the comparisons of commercially available standards.

2.5. Nanoemulsion Preparation and Characterization

The nanoemulsion was prepared by slightly modifying the reported procedures [18,19].
It was composed of 20% (w/v) active constituent, 40% (w/v) cosurfactants (1,2-propanediol/
ethanol = 1/1), 20% (w/v) PEG 40 as an emulsifier, and 20% (w/v) water. First, 20% (w/v)
of the active constituent was added to the emulsifier, and then stirred at 200 rpm to give
the oil phase. Next, the aqueous phase consisting of 1,2-propanediol, ethanol and water
were added dropwise to the oil phase mixtures. Oil and aqueous phases were mixed evenly
through stirring with a rate of 500 rpm at room temperature (26 ◦ C) for 1 h.
The particle size and polydispersity index (PDI) values of the nanoemulsions were
measured with a modular dynamic light scattering (DLS) system (Microtrac Inc, Dusseldorf,
Germany). Moreover, the structure and morphology of cinnamaldehyde-based nanoemul-
sion was characterized by transmission electron microscopy (TEM, JEM-1400 PLUS, JEOL
Ltd., Tokyo, Japan).

2.6. Repellent Longevity Bioassay

The repellent longevity of three EOs nanoemulsions and their normal solutions were
evaluated using arm-in-cage assay. Referred to the methods of Logan et al. [20], 50 female
mosquitoes which had been starved for 12 h and had not a blood meal, were used in each
cage (30 × 30 × 30 cm). Six human volunteers (male/female = 3/3) with no or little allergic
reaction to bites were selected for the trials.
Before the trials, the hands of volunteers were cleaned with water, followed by 75%
ethanol. First, 62.5 µL of 20% (w/v) EO nanoemulsion or normal solution in ethanol, was
applied to a volunteer’s hand with the exposure area of 25 cm2 . The other part of the
hand was covered. When evaluated with the nanoemulsion, the nanoemulsion without EO
served as a negative control on the other hand. When evaluated with the normal solution,
ethanol alone served as a negative control on the other hand. During the test, the control
hand was firstly inserted into the cage to confirm the aggressivity of mosquitoes. Then
the treated hand was inserted into the same cage and kept for 2 min. If any mosquitoes
landed on the hand, it meant the loss of the repellent protection. The protection time of
each volunteer was recorded, and the average time of six volunteers was calculated.
The work was carried out in accordance with the Code of Ethics of the World Medical
Association (Declaration of Helsinki) for experiments involving humans.

2.7. Statistical Analysis

The droplet sizes of nanoemulsions were expressed as mean ± standard deviation of
three replicates. In the stability study, the drop size changes of nanoemulsions at 4 ◦ C and
25 ◦ C within 28 days were calculated and processed by Graphpad Prism 8.0 (San Diego,
CA, USA). The multi-T test was used to analyze the stability between different groups, and
compare the repellent longevity between EO nanoemulsion and its normal solution. The
results were considered statistically significant when p < 0.05.
Insects 2022, 13, 1077 5 of 13

3. Results
3.1. Screen of Repellent Activity of 60 EOs Using Membrane Feeding Device
The repellent activities of 60 EOs at the dosage of 10 µg/cm2 exposed for 30 min
against female adults were screened. Results are shown in Table 1 and Table S2. The most
effective EO was cinnamon with 77% RR, which was even higher than DEET in our assays.
Seven other EOs such as marjoram, lemongrass, bay, chamomile, jasmine, peppermint2,
and thyme resulted in more than 40% RR, indicating high repellent activity. Eighteen
EOs including osmanthus, myrrh and melissa, displayed moderate repellent activity with
20–36% RR. Fourteen EOs including clove, michelia alba flower, and basil, showed poor
repellent activity with 10–19% RR. Twenty other EOs showed very low or no repellent
activity, and some EOs even had attractive action to Ae. Albopictus. DEET as a positive
control showed 59 ± 3% RR under the same conditions.

Table 1. Repelling rate of 60 EOs at 10 µg/cm2 exposed for 30 min.

RR (%) RR (%)
EO EO
Mean ± SD Mean ± SD
Cinnamon 77 ± 4% Cedarwood 16 ± 9%
Marjoram 57 ± 2% Palmarosa 15 ± 2%
Lemongrass 54 ± 3% Michelia alba leaf 15 ± 5%
Bay 51 ± 1% Lime 14 ± 3%
Chamomile 47 ± 3% Rosemary 13 ± 2%
Jasmine 44 ± 4% Parsley 13 ± 8%
Peppermint2 42 ± 10% Juniper 12 ± 6%
Thyme 41 ± 2% Lemon eucalyptus 11 ± 2%
Osmanthus 36 ± 5% Capsicum 10 ± 5%
Myrrh 33 ± 1% Angelica 9 ± 4%
Melissa 33 ± 5% Cajeput 9 ± 4%
Grapefruit 31 ± 3% Orange sweet 8 ± 3%
Sandalwood 31 ± 4% Carrot seed 8 ± 5%
May Chang 30 ± 2% Eucalyptus 6 ± 6%
Citronella ceylon 29 ± 3% Tangerine 5 ± 0%
Nutmeg 29 ± 3% Violet 3 ± 3%
Vetiver 28 ± 3% Fennel 3 ± 3%
Ay tsao 26 ± 5% Rose 3 ± 10%
Bergamot 25 ± 8% Ginger 2 ± 4%
Clary sage 25 ± 8% Ylang ylang 1 ± 2%
Petitgrain 24 ± 2% Chinese ilex 1 ± 5%
Green tea 23 ± 4% Cypress 0 ± 1%
Geranium 23 ± 4% Black pepper −1 ± 10%
Verbena 22 ± 6% Cumin −2 ± 3%
Benzoin 21 ± 2% Pine fir −5 ± 6%
Peppermint1 20 ± 3% Mandarin −6 ± 1%
Clove 19 ± 3% Rosewood −7 ± 2%
Michelia alba flower 19 ± 4% Frankincense −10 ± 10%
Basil 18 ± 7% Lemon −12 ± 7%
Patchouli 17 ± 3% DEET
59 ± 3%
Neroli 17 ± 5% (positive control)

3.2. Chemical Compositions of 8 Active EOs

There were 8 EOs with more than 40% RR. Among them, the main constituents of
lemongrass and thyme has been reported by our team [15]. The chemical compositions
of 6 other EOs were analyzed by GC-MS in this study. In addition to comparing the
retention index (RI) values with the reported data, the main components with relative area
(RA) > 5% were finally identified via comparison with commercially available standards.
In total, 21 main components (RA > 5%) were identified in the 8 active EOs.
As shown in Table 2, cinnamon EO was dominated by cinnamaldehyde (82.6%). The
main components of marjoram EO included three monoterpenes and two terpenols. The
Insects 2022, 13, 1077 6 of 13

chief component was terpinen-4-ol (32.6%), followed by α-terpineol (12.4%), γ-terpinene

(10.9%), limonene (9.4%) and p-cymene (7.1%). The constituents of bay EO were relatively
diverse. Eugenol (36.3%) and β-caryophyllene (36.7%) were the two main constituents.
Besides, cinnamaldehyde (6.4%) and diisobutyl phthalate (5.6%) also had important contri-
butions. There were only two constituents accounted for 5% in chamomile EO. The highest
constituent was diethyl phthalate (43.0%), followed by limonene (12.6%). The major con-
stituents of jasmine EO were linalool (30.2%), followed by (E)-2-hexyl-cinnamaldehyde
(25.3%) and benzyl acetate (17.1%). Menthol (30.9%) and the isomer mixtures of p-menthone
(26.8%) were the most noticeable constituents of peppermint2 EO, followed by 2,2,4,6,6-
pentamethyl-heptane (22.4%) and limonene (6.8%).

Table 2. Main chemical constituents of 8 active EOs.

Component a
No RI b RI lit. c
CZ d Oma d CC d [15] LN d OMi d JS d MHB d TV d [15]
Name CAS No.
2,2,4,6,6-pentamethyl-
1 13475-82-6 991 995 22.4
heptane
2 p-cymene 99-87-6 1025 1026 7.1 17.1
3 limonene 138-86-3 1029 1031 9.4 10.7 12.6 6.8 12.1
4 γ-terpinene 99-85-4 1059 1062 10.9
5 linalool 78-70-6 1101 1101 30.2 6.9
6 trans-p-menthone 89-80-5 1156 1154 15.9
7 benzyl acetate 140-11-4 1166 1165 17.1
8 cis-p-menthone 491-07-6 1167 1165 10.9
9 menthol 89-78-1 1175 1173 30.9
10 terpinen-4-ol 562-74-3 1179 1182 32.6
11 α-terpineol 98-55-5 1192 1189 12.4 6.9
12 neral 106-26-3 1242 1242 34.2
13 geranial 141-27-5 1271 1273 35.4
14 cinnamaldehyde 104-55-2 1272 1278 82.6 6.4
15 thymol 89-83-8 1294 1292 30.6
16 carvacrol 499-75-2 1303 1300 8.7
17 eugenol 97-53-0 1359 1356 36.3
18 β-caryophyllene 87-44-5 1426 1428 36.7
19 diethyl phthalate 84-66-2 1598 1594 43.0
(E)-2-hexyl-
20 101-86-0 1759 1749 25.3
cinnamaldehyde
21 diisobutyl phthalate 84-69-5 1875 1877 5.6
Other components 17.4 27.6 19.7 15.0 44.4 27.4 13.1 17.7
a Components are listed in the order of RI value. Only major components (RA > 5%) are listed in the table.
b Linear retention index on TG-5MS column, experimentally determined using homologous series of C -C
8 30
alkanes. c Linear retention index taken from https://webbook.nist.gov/chemistry/ (accessed on 18 July 2022).
d CZ: Cinnamomum zeylanicum (cinnamon); OMa: Origanum marjorana (marjoram); CC: Cymbopogon citratus

(lemongrass); LN: Laurus nobilis (bay); OMi: Ormenis mixta (Chamomile); JS: Jasminum sambac (Jasmine); MHB:
Mentha haplocalyx Briq (peppermint); TV: Thymus vulgaris (thyme).

3.3. Repellent Activity of 21 Main Constituents from 8 Active EOs

The repellent activity of all main constituents of the 8 active EOs was tested. The
RR of these compounds were shown in Figure 2 and Table S3. Cinnamaldehyde showed
the highest repellent activity with 82% RR, followed by citral (isomers mixture of neral
and geranial) with 65% RR, terpinen-4-ol with 60% RR, and thymol with 53% RR. Benzyl
acetate, diethyl phthalate, eugenol, diisobutyl phthalate, and β-caryophyllene performed
moderated repellent activity with >30% RR. The chemical structures of these 9 active
compounds were depicted in Figure 3.
Besides, α-terpineol, menthol, carvacrol, linalool, and limonene had some repel-
lent activity with 13-23% RR. (E)-2-hexyl-cinnamaldehyde and p-menthone (mixture of
cis- and trans-isomers) showed very low repellent activity. Three compounds including
2,2,4,6,6-pentamethyl-heptane, γ-terpinene, and p-cymene even attract mosquitoes com-
pared with the negative control. Therefore, three compounds (cinnamaldehyde, citral and
terpinen-4-ol) with higher RR than DEET, were selected as the objectives in the following
formulation study.
 activity with 13-23% RR. (E)-2-hexyl-cinnamaldehyde and p-menthone (mixture of c
and trans-isomers) showed very low repellent activity. Three compounds includi
2,2,4,6,6-pentamethyl-heptane, γ-terpinene, and p-cymene even attract mosquito
compared with the negative control. Therefore, three compounds (cinnamaldehyde, citr
Insects 2022, 13, 1077
and terpinen-4-ol) with higher RR than DEET, were selected as the objectives
7 of 13
in t
following formulation study.

Insects 2022, 13, x FOR PEER REVIEW 8 of 14

Figure 2. Repellency
Figure 2. rate of the main
Repellency rate constituents of the 8 mostofactive
of the main constituents the 8 EOs.
most active EOs.

Figure 3. Chemical structures of 9 active

active constituents
constituents from
from 88 active
active EOs.
EOs.

3.4. Characterization of Nanoemulsions of Three Active Compounds

3.4. Characterization of Nanoemulsions of Three Active Compounds
The droplet sizes of the nanoemulsions of cinnamaldehyde, citral and terpinen-4-ol
The droplet sizes of the nanoemulsions of cinnamaldehyde, citral and terpinen-4-ol
were determined using DLS. As shown in Figure 4, the average particle sizes of freshly
were determined using DLS. As shown in Figure 4, the average particle sizes of freshly
prepared samples were 50.2 ± 3.4 nm (cinnamaldehyde), 35.3 ± 1.8 nm (citral), and
prepared samples were 50.2 ± 3.4 nm (cinnamaldehyde), 35.3 ± 1.8 nm (citral), and 47.0 ±
47.0 ± 1.0 nm (terpinen-4-ol), with PDI values of 0.041, 0.057, and 0.180, respectively. In
1.0 nm (terpinen-4-ol), with PDI values of 0.041, 0.057, and 0.180, respectively. In addition,
addition, the stability of the three nanoemulsions was monitored under 4 ◦ C and 25 ◦ C.
the stability of the three nanoemulsions was monitored under 4 °C and 25 °C. As shown
As shown in Figure 5, the particle sizes of the three nanoemulsions remained stable with
in Figure 5, the particle sizes of the three nanoemulsions remained stable with storage at
storage at 4 ◦ C. No phase separation or other visual changes occurred during 28 days.
4 °C. No phase
However, separation
a relative or other of
destabilization visual changes occurred
the nanoemulsions duringat2825days.
appeared However,
◦ C after 28 daysa
relative Therefore,
storage. destabilization
theseof the nanoemulsions
nanoemulsions shouldappeared
be stored at
at 425◦ C.
°C after 28 days storage.
Therefore, these nanoemulsions should be stored at 4 °C.
Insects 2022, 13, 1077 8 of 13

Figure 4. Droplet size distribution of three nanoemulsions ((A) cinnamaldehyde; (B) citral;
(C) terpinen-4-ol).

Insects 2022, 13, x FOR PEER REVIEWFigure 5. Particle size changes of three nanoemulsions ((A) cinnamaldehyde; (B) citral; (C) terpinen-
9 of 14
4-ol) for 28 days at 4 ◦ C and 25 ◦ C (compared with 0 day, ** means p ≤ 0.01; *** means p ≤ 0.001; ****
means p ≤ 0.0001).
Moreover, the structure and morphology of the nanoemulsion of cinnamaldehyde
Moreover, the structure and morphology of the nanoemulsion of cinnamaldehyde were
were analyzed by TEM. As shown in Figure 6, phosphotungstic acid-stained
analyzed by TEM. As shown in Figure 6, phosphotungstic acid-stained cinnamaldehyde
cinnamaldehyde droplets were clearly visible and spherical. The droplet size in TEM was
droplets were clearly visible and spherical. The droplet size in TEM was similar to that
similar
measuredto that measured by DLS.
by DLS.

Figure 6.
6. Droplet
Dropletofof
cinnamaldehyde nanoemulsion
cinnamaldehyde under
nanoemulsion TEM.
under × 20,000
((A) ((A)
TEM. magnification;
× 20,000 (B) ×
magnification;
40,000 magnification)
(B) × 40,000 magnification).

3.5. Repellent
3.5. Repellent Longevity
Longevity Comparison
Comparison
The protection
The protectiontimes
timesof three nanoemulsions
of three at the at
nanoemulsions dosage
the of 500 µgofactive
dosage 500 constituent
μg active
per cm 2 against Ae. albopictus were evaluated, and compared with the same dosage of their
constituent per cm against Ae. albopictus were evaluated, and compared with the same
2

normal solutions.
dosage As shown
of their normal in TableAs
solutions. 3, except
shownforin terpinen-4-ol,
Table 3, exceptthe for
repellent longevitythe
terpinen-4-ol, of
other two nanoemulsions was significantly extended (p < 0.05). For example, the
repellent longevity of other two nanoemulsions was significantly extended (p < 0.05). For protection
time of the
example, thenanoemulsion
protection timeof of
cinnamaldehyde
the nanoemulsionwasof146 min, which was
cinnamaldehyde wasnearly 1 h longer
146 min, which
than its normal solution.
was nearly 1 h longer than its normal solution.

Table 3. Comparison between the protection times of EO nanoemulsion and its normal solution.

Average Protection Time (min)

Compounds p Value a
Normal Solution Nanoemulsion
cinnamaldehyde 94 ± 14 146 ± 19 0.004, **
Insects 2022, 13, 1077 9 of 13

Table 3. Comparison between the protection times of EO nanoemulsion and its normal solution.

Average Protection Time (min)

Compounds p Value a
Normal Solution Nanoemulsion
cinnamaldehyde 94 ± 14 146 ± 19 0.004, **
citral 49 ± 14 83 ± 15 0.017, *
terpinen-4-ol 23 ± 9 26 ± 14 0.670, ns
DEET (positive control) >360 >360
a ns (not significant) means p > 0.05, * means p < 0.05, ** means p < 0.01.

4. Discussion
4.1. Screening of Repellent Activity of 60 EOs
Nowadays, EOs have been widely used for bactericidal [21], fungicidal [22], acarici-
dal [23,24], insecticidal [25–28], medicinal and cosmetic applications [10]. Moreover, EOs
as natural insect repellents have a long usage history in China and Arab countries, thereby
being an ideal resource to discover new natural repellents [7,14,29,30]. Normally, the re-
pellent activity of EOs against mosquitoes is evaluated using an arm-in-cage assay [31,32],
which needs a certain number of qualified volunteers. Thus, extensive screening is difficult
and of low efficiency. Thanks to our modified Hemotek membrane feeding system, the
repellent activity of up to 60 EOs against Ae. albopictus was screened in this study. As
shown in Table 1, the RRs of cinnamon, marjoram and lemongrass EOs at 10 µg/cm2
exposed for 30 min were 77 ± 4%, 57 ± 2%, and 54 ± 3%, which were comparable to
that of DEET. The application of cinnamon and lemongrass EOs in the mosquito-vector
control have been widely reported [33]. Pohlit et al. [34] reviewed the patent literatures on
mosquito repellent inventions which contain plant essential oils. Cinnamon and lemon-
grass EOs were each cited in >10% of patents. Peach et al. [35] reported cinnamon bark and
lemongrass EOs are effective at spatially repelling Ae. aegypti in field settings. Our resutlts
suggested that the two EOs also had strong contact repellent activity against mosquitoes.
The reports on the repellent activity of marjoram EO against mosquitoes were relatively
few, except Kang et al. [36] reported that majoram EO showed good repellency (repellent
efficacy > 60%) at a concentration of 5 µg/cm2 against Culex pipiens pallens. Our re-
sults also supported that majoram EO had potent repellent activity against Ae. albopictus.
It is worth noting that citronella ceylon and lemon eucalyptus EOs are well-known spatial
mosquito repellents [34,37]. They repel mosquitoes via diffusing volatile odors that can
affect mosquitoes’ behavior in human host detection. However, in our assay, their con-
tact RRs were only 29% and 11%, respectively. The results suggest that there were some
differences between the contact and spatial repellent activities of the same EO, and this
difference maybe come from different action modes [38].

4.2. Identification of Active Constituents

The chemical compositions and their contents in EOs were largely impacted by the
origin, the extraction method, extraction part and other factors [39]. The efficacy of EOs de-
pends on the chemical compositions. Therefore, the active ingredients need to be identified.
The GC-MS analysis results of 8 active EOs with more than 40% RR are shown in Table 2.
There were 21 compounds accounted for more than 5% RA in these 8 active EOs. The main
constituents include terpenes, oxidized terpenes (including alcohols, aldehyde and ketone),
aldehydes, esters and others.
The repellent activities of 21 main constituents are shown in Figure 2. The high repel-
lent activity of cinnamon EO mainly came from that of cinnamaldehyde. The main active
constituent of marjoram EO was terpinen-4-ol. Citral as the mixture of neral and geranial
isomers was the main active ingredient of lemongrass EO. The high repellent activity of bay
EO was caused by the combined action of cinnamaldehyde, eugenol, diisobutyl phthalate,
and β-caryophyllene. The major active constituents of chamomile and jasmine EOs were
diethyl phthalate and benzyl acetate, respectively. Thymol had a major contribution to
Insects 2022, 13, 1077 10 of 13

the potent activity of thyme EO. In addition, carvacrol, linalool, and limonene also had
some contributions. Peppermint2 EO performed potent repellent activity in the initial
screening. However, its main constituents including p-menthone, menthol, and limonene,
only showed moderate or low repellent activity. It indicated that some minor constituents
(<5% RA) might have an important effect on the repellent activity; that is to say, a synergis-
tic phenomenon among these constituents may result in a higher bioactivity compared to
the isolated components [7].
The chemical structures of 9 active compounds with more than 30% RR are depicted
in Figure 3. Their structures were diverse, thereby it was difficult to find some common
features among them. However, their RI values were relatively big in the GC-MS analysis.
Conversely, the compounds with lower RI values such as 2,2,4,6,6-pentamethyl-heptane,
p-cymene, and γ-terpinene, displayed low repellent activity. It indicated that the physico-
chemical properties like volatility greatly affected the repellent activity [7].

4.3. Nano-Formulation Study

Some plant-derived repellents are comparable to, or even better than synthetics;
however, EO repellents tend to being short-lived in their effectiveness due to their high
volatility [7,40]. At present, the use of nanotechnology to develop new formulations is
one of the most important ways to slow the release rate and thus prolong the protection
time [37,41–43]. In our repellent assay, three compounds including cinnamaldehyde, citral,
and terpinen-4-ol, showed higher repellent activity than DEET when they were exposed
for 30 min in the mosquito cage, which suggested that they have potential for further
development as new mosquito repellents.
In our study, based on water as the aqueous phase, the active constituents (cin-
namaldehyde or citral or terpinen-4-ol) as the oil phase, PEG 40 as the surfactant, and
1,2-propanediol/ethanol = 1/1 (w/v) as the cosurfactants, the nanoemulsions were pre-
pared by a low-energy method. The nanoemulsions were characterized by particle size, PDI
value and morphological image (cinnamaldehyde), which suggests the physicochemical
characterizations of these nanoemulsions were desirable. In order to investigate the stabil-
ity of these nanoemulsions, the particle sizes were monitored for 28 days (Figure 5). The
results showed that the temperature and storage time affected the nanoemulsion stability.
The droplet sizes increased in 28 days at 25 ◦ C storage. However, no statistically significant
difference was observed in droplet sizes for 28 days at 4 ◦ C, where the p-value was >0.05.
It suggested that these nanoemulsions have good stability at 4 ◦ C, which was in agreement
with our previous study [44].
Next, the protection times of these nanoemulsions were evaluated and compared with
the normal solutions. As shown in Table 3, the protection times of the nanoemulsions of
cinnamaldehyde and citral were greatly extended. It suggests that the active constituents
were released more slowly in the nanoformulations [19]. Drapeau et al. [45] also reported
that the microemulsion of p-menthane-diol obtained an extension of the protection time
against Ae. aegypti. Nuchuchua et al. [46] reported that the nanoemulsions of citronella
oil, hairy basil oil and vetiver oil, with droplet sizes ranging from 150 to 220 nm, had
prolonged mosquito protection time. It is worth noting that the RRs of cinnamaldehyde
(82%) and citral (65%) were higher than the positive reference DEET (59%); however, their
protection times were much shorter than that of DEET, which was probably due to the high
volatility and the physicochemical instability of the two natural compounds. Our results
suggest that nano-formulations could be used to alleviate this problem. Rehman et al. [13]
reviewed the protection times of essential oils against mosquitoes. The cinnamaldehyde-
based nanoemulsion displayed a longer protection time (146 min) than most of the reported
natural repellents.

5. Conclusions
In conclusion, the mosquito repellent activity of up to 60 commercial EOs at the dosage
of 10 µg/cm2 exposed for 30 min were screened. Eight EOs including cinnamon, marjoram,
Insects 2022, 13, 1077 11 of 13

lemongrass, bay, chamomile, jasmine, peppermint2, and thyme, displayed potent repellent
activity with more than 40% RR. Twenty-one major constituents (>5% RA) in the 8 active
EOs were identified via GC-MS analysis. Cinnamaldehyde, citral and terpinen-4-ol dis-
played the highest repellent activity with more than 60% RR. Their nanoemulsions were
prepared and characterized. In the arm-in-cage assay, cinnamaldehyde- and citral-based
nanoemulsions have a prolonged mosquito protection time compared with their normal so-
lutions. This study not only discovered several leading compounds from which to develop
novel plant-derived mosquito repellents, but also suggested that the nano-formulations
could improve the duration of natural repellents. However, the repellent mechanism study
is lacking. More in-depth studies on the action mode and chemical structural modifications
remain to be explored in the future. Meanwhile, the combination of different plant-derived
repellents together will be investigated to improve their efficacies against mosquitoes.
Moreover, the nano-formulation needs to be further optimized to improve the stability of
the nanoemulsions stored at room temperature. These works are in progress in our lab, and
will be reported in due course.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/insects13121077/s1, Table S1: information of 60 EOs; Table S2:
repelling rate of 60 EOs at 10 µg/cm2 exposed for 30 min; Table S3: repelling rate of the main
constituents from 8 active EOs.
Author Contributions: W.W.: methodology, investigation, data curation, writing—original draft; Y.Y.:
investigation, data curation; Y.F.: investigation, data curation; X.R.: investigation; Y.L.: investigation;
W.L.: investigation; J.H.: investigation; L.K.: investigation; X.C.: investigation; Z.L.: data curation;
X.H.: conceptualization, writing—review & editing; L.Z.: methodology; Y.C.: methodology; Z.S.:
conceptualization, project administration, supervision, writing—review & editing. W.H.: conceptu-
alization, supervision, funding acquisition, writing—review & editing. All authors have read and
agreed to the published version of the manuscript.
Funding: The authors gratefully acknowledge Wuyi University-Hong Kong and Macao Joint Re-
search Fund (No. 2021WGALH10, Wuyi University), the Start-up Foundation for High-level Talents
of Wuyi University (No. 2017RC04, Wuyi University), Special Fund Project of Science and Tech-
nology Innovation Strategy in Guangdong Province (No. Jiangke(2018)352, Department of Science
and Technology of Guangdong Province), Jiangmen Science and Technology Project of Basic and
Theoretical Science Research (No. 2021030101400004873, Jiangmen Science and Technology Bu-
reau), and the Project of Innovative and Entrepreneurship for College Students in Wuyi University
(No. 202111349298, Wuyi University) for the financial support.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study. Written informed consent for publication was obtained from participating subjects.
Conflicts of Interest: The authors declare that they have no known competing financial interest or
personal relationships that could have appeared to influence the work reported in this paper.

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