South African Journal of Botany 163 (2023) 95 104

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Larvicidal susceptibility of essential oils from Cinnamodendron dinisii,

Callistemon viminalis and Myrcia tomentosa against Culex quinquefasciatus
(Say) (Diptera: Culicidae)

lia Assunç a
Ju ~o de Castro Oliveiraa, Israel Pereira Garciab, Eduardo Jose

Azevedo Corre
^ac,
Leonardo Henrique França de Limad, He
rica de Lima Santosc, Rafael Marlon Alves de Assisa,
Jose
Eduardo Brasil Pereira Pinto , Suzan Kelly Vilela Bertoluccia,*
a

a
Laboratory of Phytochemistry and Medicinal Plants, Department of Agriculture, Federal University of Lavras, 37203-202, Brazil
b ~o Joa
Laboratory of Cell Biochemistry, Federal University of Sa ~o del Rei, Divino

polis, MG 35501-296, Brazil
c
Minas Gerais Agricultural Research Company, EPAMIG ITAC Center, Pitangui, MG 352650-000, Brazil,
d
Exact and Biological Science Department, Federal University of Sa~o Joa~o Del Rei Campus Sete Lagoas, 35701-970, Brazil

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

Article History: Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a vector of pathogens and parasites, whose form of
Received 19 July 2023 control is the use of synthetic insecticides. However, the indiscriminate use of these products has led to the
Revised 11 September 2023 emergence of resistant populations. The objective was to evaluate the susceptibility of C. quinquefasciatus lar-
Accepted 12 October 2023
vae to essential oils (EOs) from leaves of Cinnamodendron dinisii Schwacke (CD), Callistemon viminalis (Sol. ex
Available online xxx
Gaertn.) G.Don (CV) and Myrcia tomentosa (Aubl.) DC. (MT), and the influence on biochemical parameters, in
Edited by: Dr. L. McGaw addition to the in silico potential of the most promising EO for the target acetylcholinesterase (AChE) enzyme.
Keywords:
The steam distilled EOs were analysed by gas chromatography/mass spectrometry. The EOs lethal concentra-
Biopesticides tion of 50 % (LC50) was estimated using different concentrations and probit analysis. Then, the larvae were
Biochemical analyses exposed for 1 h to the LC75 of the most promising EO, and the biochemical parameters were evaluated in
Molecular docking vitro: soluble protein concentration, reduced glutathione content, hydrogen peroxide production, lipid per-
Acetylcholinesterase oxidation levels, and AChE enzyme activity. The AChE molecular docking was also performed. The main
Filariasis vector chemical constituents in CD were pinenes, sabinene and bicyclogermacrene; in CV were a-pinene and 1,8-
cineole and in MT were (E)-caryophyllene and germacrene D. The lowest LC50 (1.43 mg. L 1) was estimated
for CD EO, although LC75 reduced all biochemical parameters and inhibited AChE activity. The docking results
for the major (bicyclogermacrene) and minor compounds (aromadendrene, a-copaene, drymenol, s - and
b-elemene) present in CD EO have high affinity with AChE. In conclusion the effects indicate the promising
potential of the CD EO for the development of larvicide against the C. quinquefasciatus.
© 2023 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a mosquito
vector of viruses and parasites that cause Japanese encephalitis, lym-
phatic filariasis, and West Nile fever in humans (WHO, 2020). Accord-
ing to the World Health Organization(WHO, 2020), approximately
17 % of all infectious diseases caused by parasites, bacteria or viruses
are transmitted by insect vectors, such as C. quinquefasciatus, and are
responsible for the deaths of 700,000 people annually. The distribu-
tion of these diseases is determined by a complex set of demographic,
environmental and social factors, with a higher incidence in tropical
and subtropical areas, mainly affecting the poorest populations
* Corresponding author.
E-mail address: suzan@ufla.br (S.K.V. Bertolucci).
(WHO, 2020). This high number of deaths could be avoided with pro-
tection measures and sanitary mobilization in the places with the
highest incidence of these vector mosquitoes.
The main method used for the control of this vector is based on
the application of synthetic insecticides (organophosphates, pyreth-
roids, and carbamates) (Namias et al., 2021). However, some studies
have already mentioned the resistance of C. quinquefasciatus popula-
tions to these products (Sougoufara et al., 2022; Yuan et al., 2019)
due to improper management, constant application and increasing
doses. In addition, these products have been considered the main
causes of human poisoning and environmental problems (Anoopku-
mar and Aneesh, 2022; Mwila et al., 2013; Nascimento and Melnyk,
2016; Puthur et al., 2021). Given this scenario, it is a concern in the
scientific community to develop new products that are safe for
human and animal health (Anoopkumar and Aneesh, 2022).

https://doi.org/10.1016/j.sajb.2023.10.026
0254-6299/© 2023 SAAB. Published by Elsevier B.V. All rights reserved.
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
Botanical products such as essential oils (OEs) have shown to be
promising candidates for the formulation of new natural insecticides
to control insect vectors (Oliveira et al., 2022; Ramzi et al., 2022;
Sharma et al., 2022). This is due to the fact that they are more advan-
tageous than synthetic insecticides because they have multitarget
mechanisms, are biodegradable and selective and have low to moder-
ate toxicity to mammals (Pavela, 2015; Rizzo et al., 2020).
Current synthetic insecticides and phytochemicals are known to
act as molecular targets of several specific and important neurochem-
icals in insect metabolism, such as acetylcholinesterase (AChE)
enzyme (Corre ^a et al., 2023). Nowadays, chemical computational
approaches have successfully demonstrated how some EOs com-
pounds act as neurochemical/neurohormonal inhibitors of mosquito
(Corre^a et al., 2023; Silva et al., 2023). The EOs from Canellaceae and
Myrtaceae families have monoterpenes and sesquiterpenes in poten-
tial to cause toxicity to insects (Inocente et al., 2019; Oliveira et al.,
2019).
Given this information, the present study was focused on deter-
mine the susceptibility of C. quinquefasciatus larvae to EOs distilled
from leaves of Cinnamodendron dinisii Schwacke - CANELLACEAE, Cal-
listemon viminalis (Sol. ex Gaertn.) G.Don and Myrcia tomentosa
(Aubl.) DC. MYRTACEAE. The larvicidal susceptibility was accessed
by in vivo and in silico assays. The hypothesis one of the three EOs can
have a strongest larvicidal susceptibility and the acetylcholinesterase
(AChE) enzyme can contribute to their toxicity.
2. Materials and methods
2.1. Plant materials and distillation of EOs
Fresh leaves were collected in the morning between August and
November 2018. The species were incorporated into the collection of
the PAMG herbarium (Empresa de Pesquisa Agropecua
ria de Minas
Gerais - EPAMIG) under the following registries: Callistemon viminalis
(59,707), Cinnamodendron dinisii (58,650) and Myrcia tomentosa
(58,644). The EOs were distilled by steam distillation using the same
methodology described by Oliveira et al. (2022).
2.2. Estimation of LC50 of essential oils
The experiments were performed according to the protocol of the
World Health Organization (WHO, 1976). The bioassays to estimate
the lethal concentration at 50 % (LC50) of the EOs of C. viminalis, C.
dinisii and M. multiflora and toxicity of the most promising EO were
performed according to the methodology described by Oliveira et al.
(2022). The EOs were diluted in 0.5 % dimethyl sulfoxide (DMSO)
aqueous solution at concentrations of 200, 100, 50, and 25 mg/mL.
The larvae of the control group were exposed only to the vehicle. For
each sample, the larvae were divided into test and control groups,
with 60 larvae for each treatment and three replicates. The larvae
were kept in a growth chamber at 26 § 1 °C with a 12-h photoperiod.
Larval mortality was monitored every 24 h during 5 days of exposure.
Probit analysis was used to determine the sublethal concentrations.
2.3. Toxicity bioassay
Toxicity bioassays were performed according to the method pro-
posed by Oliveira et al. (2022) without modifications using the lethal
concentration at 75 % (LC75) of the most promising EO. The biochemi-
cal parameters of the larvae analysed were the soluble protein con-
centration (mg/mL) according to the methodology described by
Bradford (1976), reduced glutathione (GSH) content (mg GSH/mg
protein) according to the methodology described by Jollow et al.
(1974), hydrogen peroxide (H2O2) levels expressed in mg H2O2/mg
protein according to Alexieva et al. (2001) and AChE activity
96
South African Journal of Botany 163 (2023) 95 104
expressed in nmol thiocholine/min/mg of protein according to Ell-
man et al. (1961).
2.4. Statistical analyses
A linear regression with a 95 % confidence interval was used
employing probit analysis in BioStat 5.0 (AnalystSoft, Walnut, CA,
USA), to estimate the LC50 of EOs. The effect of concentration was
considered significant if the p < 0.05. Analysis of variance (ANOVA)
of the biochemical results was performed in GraphPad Prism 5 (Sot-
matics, San Diego, CA, USA), and a p < 0.05 was considered signifi-
cant.
2.5. Essential oil chemical analysis
The chromatographic parameters for the analysis of the EOs of C.
viminalis and M. tomentosa were the same as those described by Oli-
veira et al. (2022). For the chemical analysis of the C. dinisii EO, the
injector temperature was changed to 250 °C, and the detector tem-
perature was changed to 280 °C. The initial temperature of the oven
was 50 °C and kept in isothermal conditions for 2 min 1, followed by
a temperature ramp of 4 °C min 1 to 200 °C, followed by another
temperature ramp of 15 °C min 1 to 240 °C. Isothermal conditions
were held for 15 min. All analyses were performed in triplicate. The
EOs of C. viminalis and M. tomentosa were partially analysed, where
only the peaks with a percentage of normalized area greater than 5 %
§ standard deviation (n = 3) were considered. A complete chemical
characterization was performed for the most promising EO.
For the qualitative analyses of the EOs, the operational conditions
of the gas chromatography mass spectrometry (GC‒MS) instrument
were the same as those used in the above GC analysis. The chemical
constituents of the EOs were identified by comparing their retention
indices relative to the coinjection of a standard solution of n-alkanes
(C8 C20, Sigma‒Aldrich, St. Louis, USA) and by comparing the mass
spectra with those from the library database (NIST, 2008) and litera-
ture (Adams, 2017). The retention indices were calculated using the
Van den Dool and Kratz (1963) equation and assigned after literature
confirmation (Adams, 2017).
2.6. Virtual screening and computational analysis
2.6.1. Preparation and modeling of the molecular target
To understand the relationship between the EO components of C.
dinisii and the AChE of C. quinquefasciatus, a protein sequence (Uni-
Prot ID: V5 W7Z2_CULQU) was obtained from the UniProt database
(Zhao et al., 2014). This sequence was aligned with the AChE tem-
plate of Drosophila melanogaster (PDB ID: 1DX4 chain A) (Meigen,
1830), which was found in the Protein Data Bank (PDB) (Harel et al.,
2000) using the alignment algorithm ClustalW (Larkin et al., 2007)
and ESPript3 (Robert and Gouet, 2014). Next, protein models were
generated using four homology modeling platforms: Chimaera (Yang
et al., 2012), Modeller (Webb and Sali, 2016), Swiss-Model (Water-
house et al., 2018) and Phyre2 (Kelley et al., 2015). The choice of the
best models was made considering the quality measures of the plat-
forms and the quality access results of the models submitted to
SAVES v6.0 (https://saves.mbi.ucla.edu/), PROCHECK (Laskowski
et al., 1996) and MolProbity (Williams et al., 2018).
2.6.2. Preparation of the dataset of ligands and molecular docking
A total of 29 chemical compounds present in the C. dinisii EO
(Table 2), which were subsequently obtained from the ZINC ligand
bank (Sterling and Irwin, 2015) and converted into PDBQT format
using MGLTools (Morris et al., 2009) were used in this study. The grid
box for docking the target molecule was centered on a position simi-
lar to that occupied by the crystallographic ligand of the template (9-
N-phenylmethylamino-tacrine) being determined using templates
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
and models aligned in PyMOL. The box was centered at X = 11.435,
  
Y = 43.743 and Z = 30.375 with a size of 25 A x 20 A x 20 A. The
docking procedures were performed with an exhaustiveness parame-
ter of 128 to maximize sampling with precision. For each system, the
first three poses and the best fitting pose were taken for scoring and
structural analysis. The fitting results were analysed using PyMOL
€ dinger and Delano, 2020). Virtual
and scripts written in Python (Schro
screening was performed using a serial docking simulation protocol
in AutoDock Vina 1.1.2 (Trott and Olson, 2010) to evaluate the proba-
bility that the compounds present in the C. dinisii EO bind to AChE in
C. quinquefasciatus.
2.6.3. Selection of the scoring function
To reduce the biases inherent to the scoring functions, five func-
tions were adopted to evaluate the ligands: Vina or AutoDock Vina
(Trott and Olson, 2010), Ad4_scoring (Koes et al., 2013), DkOEs_scor-
ing (Koes et al., 2013), Vinardo (Quiroga and Villarreal, 2016) and
Convex-PL (Kadukova and Grudinin, 2017). The performance of each
molecular target scoring function (AChE) was evaluated to verify
how well they separated the hit ligands known as decoys. For this
purpose, the receiver operating characteristic (ROC) (Rocha, 2022)
and enrichment factor (EF) (Huang et al., 2006) curves were used.
Initially, the true ligands were identified from studies in the litera-
ture for bioassays using the target protein expressed in a cell or in
vitro and canonical compounds, agonists or antagonists (Rocha,
2022). Only ligands with reported half maximal inhibitory concentra-
tion (IC50), half maximal effective concentration (EC50), inhibition
constant (Ki) or dissociation constant (Kd) metrics were selected as
positive controls. Then, up to 50 decoys were generated for each posi-
tive control ligand using DUD-E (Mysinger et al., 2012). All ligands in
the validation set were subjected to docking simulations using the
Vina function and scored according to all additional scoring functions,
resulting in five different classifications. Finally, these rankings were
used to construct the ROC and EF curves for each scoring function
and target combination.
3. Results and discussion
3.1. Bioassays and LC50 estimation of essential oils
Among the evaluated EOs, the C. dinisii EO showed the highest
toxicity to C. quinquefasciatus larvae in the first 24 h of the test. The
LC50 values of the EOs of C. viminalis and M. tomentosa were deter-
mined for 48 h because lethality occurred after this period. After 24 h
of exposure of C. quinquefasciatus larvae to C. dinisii EO, an LC50 of
1.43 mg/mL was observed, which is considered effective according to
the classification by Komalamisra et al. (2005) (Table 1). According to
Pavela (2015), EOs from plants with LC50 lower than 50 mg/mL can
be considered promising sources of pesticide plants.
The C. dinisii EO was potentially more effective than other botani-
cal insecticides considered promising for the control of C. quinquefas-
ciatus larvae. When comparing the LC50 observed for C. dinisii, its
Table 1
Lethal concentration (LC) of C. dinisii EO on C. quinquefasciatus larvae after 24 h of
exposure and for C. viminalis and M. tomentosa EOs after 48 h of exposure and their
respective toxicity classifications according to Komalamisra et al. (2005).
Species LC50 (mg/mL) (LCL UCL) 24 h x2
C. dinisii 1.43 (0.80 2.07) 1.38 ns
Species LC50 (mg/mL) (LCL - UCL) 48 h x2
C. viminalis 398.57 (220.33 1.966) 0.78 ns
M. tomentosa 683.90 (278.99 16.774) 0.03 ns
LC50: Concentration that killed 50 % of exposed individuals; X2 = chi-squared. LCL,
lower confidence limit; UCL, upper confidence limit
a
Classification according to Komalamisra et al. (2005).
South African Journal of Botany 163 (2023) 95 104
effectiveness was superior to the following EOs: Satureja Hortensis L.
(LC50 of 36.1 mg/mL), Thymus saturejoides Coss (LC50 of 43.6 mg/mL)
and Thymus vulgaris L. (LC50 of 32.9 mg/mL) in the Lamiaceae Marti-
nov family, as observed by Pavela (2009); Acmella oleracea (L.) R. K.
Jansen (LC50 of 42.2 mg/mL) (Benelli et al., 2019) and Baccharis dra-
cunculifolia DC. (LC50 of 34.45 mg/mL) (Alves et al., 2018) in the Aster-
aceae family; Eugenia uniflora (LC50 of 41.74 mg/mL) and Melaleuca
armillaris (LC50 of 31.52 mg/mL) in the Myrtaceae family; Schinus
molle (LC50 of 60.08 mg/mL) in the Anacardiaceae family, as deter-
mined by Oliveira et al. (2022). The C. dinisii EO can also be consid-
ered more promising than the neem oil extracted from Azadirachta
indica A. Juss seeds. (Meliaceae) tested by Okumu et al. (2007) on
Anopheles gambiae Giles, 1926 (Diptera: Culicidae) larvae. An LC50 of
10.7 mg/mL was estimated.
The EOs of C. viminalis and M. tomentosa had LC50 values that were
much higher than that of the EO of C. dinisii, and because their con-
centrations were above 200 mg/mL and below 750 mg/mL, they were
classified as having low toxicity. Nevertheless, these EOs showed a
more effective toxicity to C. quinquefasciatus larvae than others
described in the literature, such as Peumus boldos Molina (Monimia-
ceae) (LC50 of 8.214 mg/mL) (Castro et al., 2016) and Schinus terebin-
thifolia Raddi (Anacardiaceae) (LC50 of 1288.71 mg/mL) (Kweka et al.,
2011), which were also tested against C. quinquefasciatus larvae.
3.2. Biochemical responses of larvae exposed to LC75 of the EO distilled
from the leaves of C. dinisii
Considering that the screening of the three studied EOs indicated
that the C. dinisii EO was the most toxic to C. quinquefasciatus larvae,
biochemical analyses were performed with C. quinquefasciatus larvae
exposed for 1 h to a sublethal dose (LC75) of the EO of C. dinisii. The
results of the biochemical parameters (soluble protein concentration,
oxidative parameters GSH content, H2O2 levels and AChE enzyme
inhibitory activity are shown in Fig. 1.
There was a significant reduction in all biochemical parameters
measured for C. quinquefasciatus larvae when exposed to LC75 of the
C. dinisii EO (Fig. 1). However, the most affected parameter was AChE
activity (Fig. 1D).
The reduction in soluble protein concentration observed in similar
to that in other studies with botanical insecticides, such as the EOs of
Eugenia uniflora L., Melaleuca armillaris (Sol. ex Gaertn.) Sm., and Schi-
nus mole L. (Oliveira et al., 2022), which were also responsible for
altering the protein levels of C. quinquefasciatus larvae. This reduction
may be related to several factors, such as the increase in protein
demand for detoxification of the active agents of the insecticide;
reduction of protein synthesis; increase in energy demand compen-
sating for protein breakdown; formation of lipoproteins for cellular
and tissue organelle repair and incorporation of amino acids into the
polypeptide chains (Lawrence and Olorunfemi, 2019; Nath, 2002; Piri
et al., 2014; Sharma et al., 2022).
There was a significant reduction in the levels of GSH and H2O2 in
the treated group (Figs. 1B and 1C, respectively). GSH is an intracellu-
lar tripeptide important in the biotransformation and elimination of
xenobiotics and other endogenous compounds as well as in cellular
protection during oxidative stress caused by reactive oxygen species
(ROS) and other free radicals, peroxides and even heavy metals (Rai
and Saha, 2022). Nevertheless, the enzymes responsible for the
Effectivenessa
detoxification of these products may have a unique specificity, by a
class of insecticides (Strode et al., 2008), or a diverse specificity, by
Effective more than one class of insecticides (Soko et al., 2015). H2O2 is a highly
Effectivenessa
Low
deleterious reactive oxygen metabolism species because it partici-
Low pates in the reaction that produces OH_ generated in inflammatory
processes and biological dysfunctions (Ferreira and Matsubara,
1997). After its generation, H2O2 is partially eliminated by catalases,
glutathione peroxidase and thioredoxin-bound peroxidases, but
97
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
Fig. 1. Biochemical responses of Culex quinquefasciatus larvae to 1 h of exposure to LC75 of the essential oil of Cinnamodendron dinisii. A. Total protein concentration; B. reduced glu-
tathione content; C. level of hydrogen peroxide; D. acetylcholinesterase inhibitory activity. Data were evaluated by one-way ANOVA, followed by the Newman‒Keuls post hoc test,
with p < 0.05 considered statistically significant. *Significantly different from the control; p < 0.05.
because this elimination has low efficiency, much of the H2O2 is
released into the cell (Barreiros et al., 2006).
These results raised two hypotheses: 1) the decrease in H2O2
occurred because the C. dinisii EO induced a stress in the larvae and a
protective effect. Thus, GSH, when binding to H2O2, in addition to
reducing the levels of the latter, also reduces the free -SH groups in
the homogenate, resulting in lower levels of GSH in the sample. 2) C.
quinquefasciatus larvae did not develop resistance to the compounds
present in this EO. According to Rai and Saha (2022), resistance to
insecticides is developed when there is an increase in the normal
functioning of detoxifying enzymes and, consequently, a high rate of
biotransformation and elimination of these products, which prevents
them from reaching the target site in the body of the insect. Thus, the
C. dinisii EO may cause the mortality of C. quinquefasciatus larvae via
a susceptibility mechanism (Rai and Saha, 2022).
When assessing the influence of LC75 of the C. dinisii EO on the
activity of AChE, which is responsible for the hydrolysis of the neuro-
transmitter acetylcholine in cholinergic synapses, a significant reduc-
tion in this parameter was observed (Fig. 1D). This result
corroborates those described in the literature, where there are
reports that EOs from different species promoted a significant reduc-
tion in AChE in different insect species, including C. quinquefasciatus
(Oliveira et al., 2022; Souza et al., 2012). From this result, another
hypothesis is that the components of this EO inhibit this enzyme as
chemical insecticides (organophosphates and carbamates) (Cao et al.,
2020). After inhibition, there is an increase in the levels of acetylcho-
line in the synapses, causing the death of the insect by paralysis
(Alves et al., 2018; Mouche s et al., 1987) due to the impairment of
AChE function in the insect cholinergic system.
Changes in the biochemical parameters of insects are important
indicators of insect susceptibility to insecticide toxicity (Piri et al.,
98
South African Journal of Botany 163 (2023) 95 104
2014). Thus, the results of the present study indicate a susceptibility
of C. quinquefasciatus larvae to the C. dinisii EO, making it possible to
infer that the compounds present in this EO act by harming the larvae
through different mechanisms of action. However, other mechanisms
of action may be involved in the larvicidal activity of the C. dinisii EO,
and further investigations, such as histology, are needed to verify the
organs and tissues that may be involved in this activity. However,
given the biochemical responses, it can be inferred that the C. dinisii
EO has different mechanisms of action in C. quinquefasciatus larvae.
3.3. Chemical analysis of essential oils
The EO of C. dinisii had the highest content (0.50 %), followed by C.
viminalis (0.15 %) and M. tomentosa (0.04 %). The results of the chemi-
cal composition of these EOs shown in Table 2 indicate that the pres-
ence of some constituents was common among the three species,
although with different levels. These constituents include a-pinene
in C. dinisii (27.59 %) and in C. viminalis (21.72 %), 1,8-cineole in C.
viminalis (62.03 %) and C. dinisii (2.97 %), bicyclogermacrene in C. dini-
sii (11.86 %) and M. tomentosa (6.35 %) and (E) -caryophyllene in C.
dinisii (3.75) and M. tomentosa (15.05).
The main composition of the distilled EO of C. viminalis leaves con-
sists of two major compounds: 1,8-cineole (62.03 %) and a-pinene
(21.72 %). These data corroborate those described in the literature
(Mahmoud et al., 2019; Sarma et al., 2019). Other studies have shown
that this EO had a 1,8-cineole content above 60 % and a variable con-
tent between 0.38 % and 12.34 % for a-pinene (Ngom et al., 2018;
Oyedeji et al., 2009; Romeilah et al., 2016). Pires et al. (2013) did not
identify a-pinene in this EO. On the other hand, Sarma et al. (2019)
observed an a-pinene content (32.6 %) higher than that of 1,8-cineole
(24.9 %).
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al. South African Journal of Botany 163 (2023) 95 104

Table 2
Chemical composition of the essential oils of C. viminalis, C. dinisii and M. tomentosa.

N
digo ZINCa
Co Compounds IRb Lit. (%)

C. viminalis C. dinisii M. tomentosa

1 ZINC2035755 a-tujene 925 924 0.94 § 0.01

2 ZINC967579 a-pinene 932 932 21.72 § 2.3 27.59 § 0.17
3 ZINC1673034 camphene 946 946 0.61 § 0.00
4 ZINC1599725 sabinene 972 969 12.93 § 0.12
5 ZINC967582 b-pinene 975 974 15.06 § 0.14
6 ZINC1530331 myrcene 991 988 2.04 § 0.00
7 ZINC967593 a-terpinene 1015 1014 0.19 § 0.00
8 ZINC968246 p-cymen 1023 1020 1.47 § 0.00
9 ZINC86034086 sylvestrene 1027 1025 1.95 § 0.00
10 ZINC967566 1,8-cineole 1030 1026 62.03 § 3.4 2.97 § 0.00
11 ZINC1531619 Z-b-ocimene 1038 1032 2.58 § 0.00
12 ZINC1531618 E-b-ocimene 1048 1044 2.02 § 0.01
13 ZINC967594 g -terpinene 1058 1054 0.47 § 0.00
14 ZINC968225 terpinolene 1087 1086 0.17 § 0.00
15 ZIN391936 (E)-hydrate sabinene 1100 1098 0.63 § 0.01
16 ni 1165 0.15 § 0.01
17 ZINC4262096 terpine-4-ol 1177 1174 1.06 § 0.01
18 ni 1209 0.21 § 0.01
19 ZINC1529208 neral 1241 1235 0.58 § 0.01
20 geranial 1271 1264 1.16 § 0.00
21 ni 1286 0.19 § 0.03
22 ZINC59587259 d-elemene 1338 1335 0.34 § 0.02
23 ZINC391936 a-terpinyl acetate 1416 1346 0.78 § 0.01
24 ZINC57988166 a-copaene 1436 1374 0.17 § 0.00
25 ZINC95098821 b-elemene 1392 1389 0.38 § 0.00
26 ZINC88234282 (E)-caryophyllene 1418 1417 3.75 § 0.00 15.05 § 0.10
27 ZINC49708061 aromadendrene 1441 1439 0.35 § 0.11
28 ZINC253666771 a-humulene 1456 1452 0.24 § § 0.00
29 ZINC59586886 E-b-farnesene 1458 1454 0.41 § 0.00
30 germacrene D 1480 1484 17.62 § 0.20
31 ZINC101785466 bicyclogermacrene 1499 1500 11.86 § 0.11 6.35 § 0.10
32 ni 1510 0.26 § 0.08
33 ni 1526 0.28 § 0.01
34 ni 1565 0.77 § 0.04
35 d-cadinene 1577 1522 6.42 § 0.00
36 ZINC33830103 caryophyllene oxide 1581 1582 2.81§0.03
37 ni 1587 0.71 § 0.01
38 ZINC6031137 viridiflorol 1595 1592 0.11 § 0.00
39 ni 1642 0.62 § 0.05
40 ni 1668 0.24 § 0.00
41 ni 1675 0.23 § 0.00
42 ni 1683 0.12 § 0.00
43 ZINC31160272 drimenol 1767 1766 0.21 § 0.00
TOTAL 83.75 % 99.61 % 45.44 %
a
ZINC code available at https://zinc.docking.org/.
b
Retention index relative to n-alkanes series (C8 C20) in HP-5MS column in elution order. SD: standard deviation (n = 3). ni: not identified.

The EO distilled from M. tomentosa leaves had the following major
constituents: germacrene D (17.62 %), (E)-caryophyllene (15.05 %),
d-cadinene (6.42 %) and bicyclogermacrene (6, 35 %). According to
the literature, the main composition of the M. tomentosa EO exhibits
chemovariability according to the region collected (Borges et al.,
2013). In addition, in this study, the authors found that some com-
pounds ranged from undetected to high concentrations, such as
b-caryophyllene (0.80 to 12.46 %), g -muurolene (undetected to
40.16 %), bicyclogermacrene (not detected to 17.32 %), d-amorphene
(not detected to 18.83 %), spathulenol (not detected to 20.09 %), glob-
ulol (not detected to 67.49 %) and (2E, 6E)) -methyl farnesoate (not
detected to 60.69 %) (Borges et al., 2013).
Chemical analysis of 99.89 % of the total composition of the C. dini-
sii EO indicated the presence of 42 constituents. Of these, 31 constitu-
ents (96.11 % of the total chemical composition) were identified, and
11 (3.78 %) were not identified. The major constituents were
a-pinene (27.59 %), b-pinene (15.06 %), sabinene (12.93 %) and bicy-
clogermacrene (11.86 %). The main composition of this EO corrobo-
rate the study by Loura et al. (2016).
3.4. Virtual screening and computational analysis
To evaluate the relationship between the chemical composition of
the C. dinisii EO and its AChE-reducing activity, this enzyme was cho-
sen as the biological target for the molecular docking study.
3.5. Similarity between the ache of the mosquito C. quinquefasciatus
and the fly cochliomyia hominivorax
The similarity between the AChE model of the C. quinquefasciatus
mosquito and the AChE model of the C. hominivorax fly was 50.65 %.
Given the similarity between the models and the conservation of the
AChE catalytic site between these two species, the same molecular
archetypes that were determined in the unpublished study by Corre ^a
et al. (2023) were considered. Natural compounds from the ligand
bank SistemamatX were docked in the AChE model of the fly Cochlio-
myia hominivorax. Thus, the clusters with the molecular signatures of
the archetypes for the AChE model of the mosquito C. quinquefascia-
tus were plotted.

99
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
Fig. 2. Alignment of the Culex quinquefasciatus AChE model with the 1DX4 template of the Drosophila AChE PDB of the model generated in Modeller 9.25. The Culex quinquefasciatus
model is shown in blue, and the Drosophila crystallographic template is shown in red. The catalytic residues of AChE according to Zhao et al. (2014) and da Silva, de Carvalho & de
Azeredo Espin (2011) are represented by green sticks, and the 9-N-phenylmethylamino-tacrine crystallographic ligand is shown in orange.
The models generated for the AChE of C. quinquefasciatus with the
1DX4 template of the Drosophila melaongaster AChE PDB showed low
root mean square deviation (RMSD), with values ranging from 0.00
to 0.394 (Fig. 2).
The final ranking of the ligands according to the order of highest
to lowest affinity was performed using the mean of the highest affin-
ity of the four models (four affinity values) and the mean of the three
highest affinities of the four models (12 affinity values). The literature
describes that when the root mean square deviation (RMSD) values

range from 0 to 2 A, the simulations can be considered validated (Vel-
loso et al., 2020; Wang et al., 2003). Thus, the results of this alignment
corroborated with those described, as the RMSD was 0.192. In addi-
tion, it can be considered that this AChE model was satisfactory
because according to Wang et al. (2003) and Velloso et al. (2020), the
lower the RMSD is, the greater the structural similarity and the better
the model.
Fig. 3. Correlation of the euclidean distances in the z scores of the compounds present
in the Cinnamodendron dinisii EO.
100
South African Journal of Botany 163 (2023) 95 104
After obtaining the AChE model, a library of ligands consisting of
29 chemical constituents of the EO of C. dinisii was created, and the
ZINC codes of these compounds are shown in Table 2. Next, we pro-
ceeded with virtual screening to search for the best pose between the
ligands and the target molecule to evaluate the probability of the
compounds present in the C. dinisii EO binding to C. quinquefasciatus
AChE. The positive controls and the decoys generated were recorded
in the 4 AChE models generated from C. quinquefasciatus. The data
were tabulated considering the average of the highest scores
obtained, with the ordering of the ligands of greater affinity to the
receptor for the lower affinity to the receiver, and the receiver oper-
ating characteristic (ROC) and the enrichment factor (EF) curves were
constructed. These curves showed the recovery during model dock-
ing of the ligands of compounds that are truly or experimentally
determined to be reversible AChE inhibitors. Considering the results,
the scoring functions chosen were VINA, AD4 and DKOES, as they
showed an area under the curve (AUC) ranging from 0.490 to 0.709.
In addition, the recovery of the enrichment curve close to 1 %
(log1 = 0) and the maximum enrichment factor (maxEF) values were
taken into account. These values, which represent the maximum of
the factor curve, i.e., the recovery of positive controls, ranged from
5.25 to 14.77. The maxEF value is considered satisfactory when it is
closer to 1 %. Thus, the VINA, AD4 and DKOES functions were chosen
using the mean of the first three affinity calculations (12 values).
After the selection of the scoring functions and construction of the
ROC and EF curves, postprocessing of the molecular docking results
was performed. Thus, after comparing the clusters containing chemi-
cal structures of natural plant compounds obtained from the Sistemat
X database (Costa et al., 2021) related to the AChE of Chrysomya albi-
ceps (Corre ^a et al., 2023), followed by C-means clustering using the
centroid and Euclidean distance to allocate the C. dinisii EO com-
pounds, it was possible to compare the similarity of the molecules
using different metrics to obtain the fingerprints (Santana et al.,
2021) and validate the degree of influence of the compounds present
in this EO on the AChE of C. quinquefasciatus.
Figs. 3 and 4 show the correlation of the Euclidean distances of the
compounds present in the C. dinisii EO in relation to AChE and the
mean rank of the 3 score functions, respectively.
According to the prediction, using the Euclidean distances in the z
scores of the type 3 protocol, the major (bicyclogermacrene) and
minor (aromadendrene, a-copaene, drimenol, s -elemene and b-ele-
mene) compounds present in the EO of C. dinisii were the compounds
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
Fig. 4. Mean rank of the three selected score functions.
with the highest affinity with AChE, i.e., at the top of the < 5 type
ranking.
As more general approach, the molecular docking energies of all
ligands with AChE were analysed and compared (Fig. 5).
It was found that those compounds that showed a low ranking
have a high affinity for AChE. Among these compounds, one com-
pound was classified as major (bicyclogermacrene) and five com-
pounds were classified as minor (aromadendrene, a-copaene,
drimenol, s -elemene and b-elemene) according to their percentage
in the total composition of C. dinisii. Thus, as the results of this in silico
study corroborate those previously described, it is inferred that the
Fig. 5. Classification of Cinnamodendron dinisii essential oil compounds in ascending order of greater interaction of the ligand with AChE according to the scores of the selected func-
tions.
101
South African Journal of Botany 163 (2023) 95 104
aforementioned components are responsible for AChE inhibition, act-
ing as synthetic insecticides (organophosphates and carbamates)
(Cao et al., 2020).
Analysing the density maps (Fig. 6) of the crystallographic struc-
ture of the AChE of D. melanogaster experimentally determined by X-
ray crystallography and superimposed on the best pose of these 5
ligands in the AChE model of C. quinquefasciatus, it is possible to per-
ceive a better occupation of the density space by the compounds aro-
madendrene and drimenol, as shown in Fig. 6A and B, respectively.
Thus, we can attribute AChE inhibition to the five C. dinisii EO com-
pounds (aromadendrene, drimenol, a-copaene, s -elemene, b-ele-
mene and bicyclogermacrene).
In addition, it is inferred that the aforementioned minor and
major compounds act synergistically. These interactions are complex,
and although these combinations provide a basis for the formulation
and development of new botanical insecticides (Kanda et al., 2017), it
should be noted that due to the prediction, it was pointed out that
the minority compounds were those that present high affinity with
AChE. It is important to investigate formulations containing the C.
dinisii EO and not only a combination of some of its molecules.
Studies have shown that EOs rich in compounds such as a- and
b-pinene, myrcene, germacrene B and D, and 1,8-cineole, among
others, have larvicidal activity against larvae and various disease vec-
tors (Oliveira et al., 2022; Ramzi et al., 2022; Yuan et al., 2019).
Another major compound present in the C. dinisii EO is bicyclogerma-
crene (11.86 %), which also had its larvicidal potential confirmed by
tests with Aedes aegypti larvae (Santos et al., 2006), Anopheles subpic-
tus (Grassi, 1899), Aedes albopictus (Skuse, 1894) and Culex tritaenio-
rhynchus (Giles, 1901) (Govindarajan and Benelli, 2016). Thus, the
effective activity of the C. dinisii EO against C. quinquefasciatus larvae
can be attributed to the chemical compounds of this EO.
In general, the biological activities of EOs are associated with the
synergism of their constituents. Thus, the synergism between mole-
cules is considered important because it prevents the emergence of
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
 
Fig. 6. Mesh density maps (red = 1 A, blue = 2 A) demonstrating how the Cinnamodendron dinisii EO ligands occupy the space of the crystallographic ligand. AChE model of Culex
quinquefasciatus (represented as pink sticks). A: aromadendrene (in cyan); B: drimenol (in green); C: a-copaene (in pink); D: b-elemene (in yellow); E: d-elemene (in navy blue); F:
bicyclogermacrene (in 40 % ash); tryptophan residue W82 (in blue).
resistant insect populations, making the botanical insecticide a prom-
ising effective and ecologically correct pesticide (Lourenço et al.,
2018). In addition, according to Lomonaco et al. (2009), when a dou-
ble bond of a given compound undergoes hydrogenation, the lipo-
philic character of these compounds is reduced, and their passage
through the cuticle of the larvae is hampered. Thus, the presence of
double bonds in the compounds is important for larvicidal activity.
Several mechanisms of action have been described for EOs, e.g.,
AChE inhibition, octopamine receptor activation, gamma-aminobuty-
ric acid (GABA) receptor binding, and P450 cytochrome inhibition
(Jankowska et al., 2018; Oliveira et al., 2022). However, because EOs
have a complex composition of active substances, in general, they
can act through a combination of mechanisms of action due to the
synergism of these molecules (Benelli et al., 2017; Oliveira et al.,
2022) and alter cellular biochemical processes (Ryan and Byrne,
1988). Furthermore, according to Pavela (2015), the lipophilic charac-
teristics of EOs modulate larvicidal activity.
Because the C. dinisii EO inhibited AChE and its chemical constitu-
ents indicated a high affinity for this enzyme as well as organophos-
phate and carbamate insecticides (Cao et al., 2020), further
investigations of the physiological selectivity of this EO for nontarget
organisms are necessary. This is mandatory for the development of a
new insecticide to prevent future formulations containing this EO
from causing environmental disorders and human and animal health
problems, as synthetic insecticides have caused in recent years
(Anoopkumar and Aneesh, 2022; Puthur et al., 2021).
102
South African Journal of Botany 163 (2023) 95 104
Our research hypothesis about differences in the larvicidal suscep-
tibility of the three EOs was assured. Among the three EOs evaluated,
C. dinisii leaves EO has the strongest larvicidal activity against C. quin-
quefasciatus. Both in vivo and in silico assays contribute to practically
and quickly explored that six terpenes present in the C. dinisii EO
were responsible for inhibiting the AChE enzyme, a well-known
molecular target of carbamate and organophosphate insecticides.
Our results bring new insights about a promising natural source of
insecticides, which would offer an eco-friendly, human safe and
effective to control the incidence of this vector mosquito along the
tropical and subtropical areas.
4. Conclusion
This discovery contributes in an unprecedented way to the larvi-
cidal activity against C. quinquefasciatus and indicates that the C. dini-
sii EO is the most promising among the EOs evaluated. The major
constituent and five other minor constituents of the C. dinisii EO have
a high affinity with AChE, as determined by the in silico tool. The C.
dinisii EO negatively affects the biochemical parameters of the larvae
of this insect. However, further studies are still needed, as there are
still many issues to be analysed to clarify the mechanisms, advan-
tages and limitations in the use of products of botanical origin in vec-
tor control. Histological evaluation and physiological selectivity
assays of this EO were performed to evaluate its impact on nontarget
species. This research gave valuable biological and structural insights
J.A.d.C. Oliveira, I.P. Garcia, E.J.A. Corr^ea et al.
into the larvicidal activity of volatile terpenes against C. quinquefas-
ciatus.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
CRediT authorship contribution statement

lia Assunç a
Ju ~o de Castro Oliveira: Writing review & editing,
Investigation. Israel Pereira Garcia: Investigation, Data curation, For-
mal analysis, Writing review & editing. Eduardo Jose
Azevedo
Corre^a: Data curation, Formal analysis, Investigation, Writing –
review & editing. Leonardo Henrique França de Lima: Investigation,
Data curation, Formal analysis, Writing review & editing. He
rica de
Lima Santos: Investigation, Data curation, Formal analysis, Writing
review & editing. Rafael Marlon Alves de Assis: Visualization, Writ-
ing review & editing, Software. Jose
Eduardo Brasil Pereira Pinto:
Writing review & editing. Suzan Kelly Vilela Bertolucci: Conceptu-
alization, Project administration, Resources, Writing review &
editing.
Acknowledgments
The researchers are grateful the National Council for Scientific and
Technological Development (CNPq), the Coordination for the
Improvement of Higher Education Personnel (CAPES: Finance Code
001) and the Minas Gerais State Research Foundation (FAPEMIG) for
financial support. In special, the authors would like to thank Professor
Stenio Alves Nunes (in memory) for his dedication and contribution.
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