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J. of Plant Protection and Pathology, Mansoura Univ., Vol 12 (1):11 - 17, 2021
Journal of Plant Protection and Pathology
Journal homepage: www.jppp.mans.edu.eg
Available online at: www.jppp.journals.ekb.eg
* Corresponding author.
E-mail address: ashashem2014@gmail.com
DOI: 10.21608/jppp.2021.149515
Nanoemulsions of Chamomile and Cumin Essential Oils: As an Alternative
Bio-rational Control Approach against the Red Flour Beetle, Tribolium
castaneum
Hashem, A. S. 1* and Marwa M. Ramadan2
1 Stored Product Pests Research Department, Plant Protection Research Institute, Agricultural Research Center, Sakha, Kafr El-
Sheikh, Egypt
2Economic Entomology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt
Cross Mark
ABSTRACT
Essential oil (EO) nanoemulsion is a new approach to formulate and convey insecticides and to
minimize some of the common shortcomings associated with the conventional formulations of synthetic
insecticides and also of essential oils. The aim of the present was to develop an oil-in-water (O/W)
nanoemulsion of the essential oils of chamomile (Matricaria chamomilla L.) and cumin (Cuminum cyminum
L.), and assess their lethal and sublethal toxicity to the red flour beetle Tribolium castaneum (Hersbt). The
nanoemulsions of EO were characterized by droplet sizes of 341.4 and 387.1 nm for the chamomile and
cumin, respectively. The polydispersivity (PDI), viscosity (cP), zeta potential (mV) and conductivity (mS/cm)
of the nanoemulsions were also characterized. The cumin nanoemulsion exhibited higher lethal toxicity to the
flour beetle, besides of compromising the insect weight gain while impairing their food consumption and
conversion rate in sublethal exposure. Cumin EO nanoemulstion also sparked anti-feeding activity, reduced
progeny production and prevented grain weight loss by the red flour beetle indicating its potential for stored
product protection.
Keywords: Essential oils; Nanopesticides; Stored product beetles; Insecticidal activity
INTRODUCTION
Food production is key to food security and thus
issues concerning pest management are crucial. This has
become more so as the world is trying to produce more food
to feed a growing population (Hagstrum and Phillips, 2017).
One of the main ways to increase food production is to
improve pest control and management minimizing potential
adverse environmental and human health impacts (Lusk and
McCluskey 2018). Nowdays alternative control methods are
received more attention than conventional synthetic
pesticides (Athanassiou et al., 2018). Among these methods,
biopesticides, and more particularly naturally occurring
insecticides that obtained from plants, are targets of particular
attention (Walia et al., 2017).
Large quantities of cereal crops are yearly lost in
temperate (5–10%) and tropical regions (> 20%) by pest
infestations in the stone (Rajendran, 2002). The red flour
beetle, Tribolium castaneum Herbst (Coleoptera:
Tenebrionidae), is one of the most cerious insects of stored
grains. Both adults and larvae of the red flour beetle feed on a
wide variety of stored products, including milled cereal
products and causing extensive losses in both the quality and
quantity of these products (Rees, 2004). Besides losses due to
grain consumption, the infestations also resulted in elevation
of temperature and moisture conditions leading to mold
development, including that of toxigenic species (Magan et
al., 2003).
The necessary management of the red flour beetle is
usually achieved with the use of traditional grain protectants
and fumigants, which are cost-effective in many storage
systems and also against several insect pest species (Boyer et
al., 2012). However, the use of these compounds does impose
intrinsic risks to environmental and human health (Abbassy
et al., 2014). In addition, stored-grain insects and the red flour
beetle, in particular exhibit widespread problems of
insecticide resistance (Opit et al., 2012). Therefore, the
demand for alternative control methods with improved safety
profile is in high demand for stored product protection.
In recent years, natural plant products have been a
focus of intensive research as environmentally safer pest
control materials (Saad et al., 2018). Further, their low
mammalian toxicity, and insect selectivity and resistance
profiles are promising frequently requiring longer time and
larger populations for the development of insecticide
resistance (Jindal et al., 2013). Essential oils from higher
plants are considered one of the most efficient alternative bio-
rational control methods against stored product insects
(Regnault-Roger et al., 2012). These natural compounds are
active against a wide range of pests like mites, insects,
nematodes, weeds, and fungi (Çalmaşur et al., 2006). They
also exhibit a wide range of distinct modes of action leading
to divergent expression of toxicity (Mossa, 2016). These
characteristics reinforce the perception of the potential of
essential oils as pest management tools, particularly for
organic farming (Adil et al., 2015). However, the field use of
essential oils requires suitable formulations to allow the
expression of their potential (Benelli et al., 2017).
Nanotechnology is currently considered a novel
approach in diverse fields of research and also has potential
Hashem, A. S. and Marwa M. Ramadan
for the development of improved insecticide formulations
through exhibiting small particle sizes ranging from 50 to 200
nm (Tadros et al., 2004). This interest in nanotechnology for
the development of pesticide formulations, including
nanoemulsions, is due to their promising applications in
material science, medicine, pharmacology and agriculture
(Singh et al., 2018). Nano-size materials show general and
biological properties that include large increase of
surface/area and hence increased ability to reach the target
site, in addition to fast penetration and selective accumulation
in various types of cells (Hashem et al., 2018; Madhyastha
and Daima, 2018). A nanoemulsion is an example of these
nanoformulations, which exhibits droplet sizes ranging from
20 to 200 nm (Sugumar et al., 2014).
An alleged advantage of nanoemulsions is their
solubility of natural pesticides such as essential oils without
using any organic solvent, which increases their
environmental safety profile (Wang et al., 2007). Therefore,
essential oil nanoemulsions seem to solve the inconvenient of
the rather frequent low water solubility of (organic) pesticides
(Fernandes et al., 2014). Here the development of
nanoemulsions of essential oils from two plant species - the
German chamomile and the cumin, which were prepared as
oil-in-water two-phase nanoemulsion formulations were
reported. The chemical components of the essential oils, the
nanoemulsion characterization and their lethal and sublethal
toxicity to the red flour beetle were also assessed and here
reported.
MATERIALS AND METHODS
Insects The red flour beetles were obtained from stock
colonies that maintained at the laboratory of Stored Product
Insects of the Sakha Agricultural Research Station,
Agriculture Research Center (ARC), Egypt. Cultures were
kept at 28O ± 2O C, 65 ± 5% R.H., and 16: 8 (L:D)
photoperiod. The insects were reared in 250 ml jars and the
adults were obtained by sieving the feeding substrate. The
adult insects (unsexed; 4-7 days old) that sieved out of the
stock colony were used in the experiments on the following
day.
Essential oils
Essential oils of German chamomile (Matricaria
chamomilla L.) and cumin (Cuminum cyminum L.) are
available in Egypt and were provided, as a gift, from Hashem
Brothers Company for Essential Oils and Aromatic Products
(Kafr-Elsohby, Kalyoubeya, Egypt).
Chemical constituents of the essential oils
Chemical components of essential oils were identified
with gas chromatography-mass spectrometry (GC/MS) using
the HP5890 system with a HP column (60 meter X 0.25
millimeter, 0.25 μm film thickess) (Hewlett Packard, Palo
Alto, CA, USA). The oils were detected using flame
ionization detector (FID); nitrogen and hydrogen formed the
stationary phase. The initial temperature was 60 °C, the
maximum temperature was 250 °C, and the injector
temperature was 240 °C. The relative amounts of the oil
components were calculated from the total area of the
detected peak obtained using the equipment. All of the steps
of sample preparation, extraction and analysis procedures
were carried out in the Laboratory of the Hashem Brothers
Company (Abdel Moneim Riad St., Giza, Egypt).
Chemicals
Polysorbate 80 (Tween 80) and ethanol were obtained
from El-Gomhouria for Trading Chemicals and Medical
Appliances (Egypt).
Nanoemulsion preparation
Oil-in-water nanoemulsions of the two oils (14%)
were prepared according to Hamouda et al. (1999), which
were further detailed by Joe et al. (2012). Tween 80 was used
as a non-ionic surfactant. The oil phase of the nanoemulsion
consisted of the selected essential oil representing 14% of the
total emulsion; ethanol (3%) and biosurfactant (Surfactin,
Tween 80; final concentration 3%) representing 20% (v/v) of
the emulsion (Hashem et al., 2018). The oil phase was mixed
and kept for 1 h at 86 ◦C, and subsequently mixed with
distilled water (80% v/v), kept for 3 min and finally
centrifuged at 10,000 rpm for 15 min.
Nanoemulsion characterization
The nanoemulsion physicochemical properties the
average droplet size, viscosity (cP), polydispersivity (PDI),
zeta Potential (mV) and conductivity (mS/cm) were
characterized. The droplet size and viscosity were determined
by the dynamic laser scattering method (Zetasizer Nano
ZS90) (Jun et al., 2015). The zeta potential and
polydispersivity index were determined by photon correlation
spectroscopy using the kit ZetaPlus (Zhermack, Badia
Polesine, Italy), (Arancibia et al., 2017). All characterization
analyses were performed at the Electron Microscopy Unit of
the Faculty of Agriculture at Mansoura University (Egypt).
Nanoemulsion toxicity
Acute concentration-mortality bioassay
Acute toxicity of both nanoemulsions was determined
by concentration-mortality bioassay where 1 mL of the
nanoemulsion at the concentrations of 0, 25, 50, 75 and 100
mg/mL were applied on 20 g cracked wheat grains and left to
dry for 20 min. The wheat grains treated with the
nanoemulsion were placed in 50 mL glass jars, which
subsequently received 10 non-sexed adult flour beetles (7-14
days old). Insect mortality was recorded after 96 h exposure.
All bioassays were repeated three times.
Time and concentration-dependent mortality
asssessments
Adult flour beetles were exposed to cracked wheat
grains treated with EO nanoemulsion as previously described
for the acute toxicity bioassays. However, the bioassays were
performed under different exposure times allowing mortality
assessments at 3, 6, 9 and 12 days after treatment. Again, each
combination of essential oil and concentration was replicated
three times.
Nutritional indices
The insects and the cracked wheat grains were used as
rearing substrate. They were initially weighted before the
insect release in each experimental unit following the
treatments indicated above. The insects were left on the
cracked wheat for eight consecutive days, after which adult
mortality and insect and grain weight were estimated. The
nutritional indices were calculated following Farrar et al.
(1989), as follow: relative growth rate (RGR) = (A - B)/B ×
day -1; relative consumption rate (RCR) = D/B × day -1;
conversion efficiency of ingested food (ECI) (%) = (RGR) /
(RCR) × 100; and feeding deterrence index (FDI) (%) = (C -
J. of Plant Protection and Pathology, Mansoura Univ., Vol 12 (1), January, 2021
T)/C × 100; where A = weight of live insects on the
investigated day (mg) / number of live insects on the
investigated day, B = initial weight of insects (mg) / initial
number of insects, D = biomass ingested (mg) / number of
live insects on the investigated day, C = food consumption in
control and T = food consumption of treatment.
Progeny production and grain loss
The adult insects released on the cracked wheat
grains, as described above, were removed after eight days,
and the grains were maintained for eight additional weeks
under the same environmental conditions as previously
described. After this period, the F1 progeny emergence was
recorded avoiding the overlapping of generations. The grain
loss caused by the insects was also recorded at this time.
Statistical analyses
Toxicity of the EO nanoemulsions was estimated by
probit methods using the software PcProbit (LdP Line,
available at http://www.ehabsoft.com/ldpline/) and following
Finney (1971). The remaining results were subjected to
regression analyses with concentration and time, as
independent variables (adult mortality), or only concentration
(other results), and using the curve fitting procedure of the
softwares TableCurve 3D (for adult mortality) and
TableCurve 2D (remaining results) (Systat, San Jose, CA,
USA). The regression models were selected from the simplest
(linear) to more complex models based on parsimony, F-
values (and error estimates), and steep increase/decrease in
R2 with model complexity.
RESULTS AND DISCUSSION
Results
Chemical composition of the essential oils
The results of Gas Chromatography/ Mass
Spectrometry (GC/MS) analyses of the tested oils that
obtained by hydrodistillation are summarized in Table 1.
The main compounds (>1%) of the tested oils were identified
by matching their spectra with those available in the mass
spectra digital library of the GC/MS. Thus, the main
components recognized were: bisabolol oxide A (40.54%),
7,11-dimethyl-3-methylene (17.01%), and Bisabolol oxide B
(7.43%) in the chamomile EO; and γ-terpinene (15.76%),
benzene methanol (11.32%) and beta-pinene (10.37%) in the
cumin EO.
Table 1. Composition of the chemical components of the
essential oils of chamomile and cumin used as
nanoemulsion.
Chemical
component
of essential oil
Chamomile (flowers)
Cumin (seeds)
Retention time
(min)
Concentration
(%)
Retention time
(min)
Concentration
(%)
7,11-Dimethyl-3-
methylene
28.58
17.01
-
-
Germacrene-D
29.35
1.90
-
-
Germacrene-B
29.94
1.26
-
-
3,7,11-Trimethyle
30.45
1.14
-
-
5,8-Dimethylisoquinoline
30.64
1.11
-
-
Alpha-bisabolol
36.52
6.43
-
-
Bisabolol oxide B
35.28
7.43
-
-
Chamazulen
39.23
3.52
-
-
Bisabolol oxide A
40.52
40.54
-
-
Lend-in-dicycloether
44.87
6.32
-
-
Benzene methanol
-
-
10.94
11.32
γ-Terpinene
-
-
11.23
15.76
Beta Pinene
-
-
8.56
10.37
P- cymine
-
-
12.05
7.45
1-pPhenil-1-butanol
-
-
8.25
6.45
Nanoemulsion characterization
The characterization of both nanoemulsions, from
chamomile and cumin essential oils, are shown in Table (2).
The average size and conductivity of chamomile and cumin
NE were 341.4 nm and 0.033 mS/cm for the former, while it
were 387.1 nm and 0.072 mS/cm for cumin NE. Also, the
zeta potential was -3.2 ± 4.28 for the chamomile NE and -10.1
± 4.08 for the cumin NE. However, the polydispersivity index
(PDI) value was slightly higher for the cumin NE (0.628) than
for the chamomile NE (0.069), both of which exhibited
similarly low viscosity (0.8872 cP), which may be due to the
usually low oil content of nanoemulsions.
Table 2. Physical characteristics of the nanoemulsion formulations.
Source of oil
Z-Average (nm)
Poly dispersivity (PDI)
Viscosity (cP)
Zeta Potential (mV) ± SE
Conductivity (mS/cm)
Chamomile
341.4
0.069
0.887
-3.2 ± 4.28
0.033
Cumin
387.1
0.628
0.887
-10.1 ± 4.08
0.072
Short-term contact toxicity
The NE of both essential oils were subjected to
concentration-mortality bioassays with 96 hours exposure of
adult flour beetles to assess their acute contact toxicity to these
insects. The results of probit analyses indicated suitability of
this model for the concentration-mortality curves of both NE
(high 2-values and P > 0.05) (Table 3)..
Table 3. Relative toxicity of nanoemulsion of the essential
oils of chamomile and cumin against the red
flour beetle Tribolium castaneum
Essential
oil
Slope
(± SE)
LC50 (95% FL) (mg/ml)
2
P
Chamomile
1.69
685.96 (548.77-857.45)
4.37
0.39
Cumin
4.68
136.25 (108.9-170.3)
3.43
0.44
The cumin NE was 5x more toxic to adult red flour
beetles than the chamomile NE based on the estimated LC50s
(Table 3). The higher slope observed with cumin NE also
indicated a higher homogeneity of response to this
formulation when compared with the chamomile NE
Time and concentration-dependent mortality
Extended exposure of adult red flour beetles to
cracked wheat grains treated with increasing concentrations
of either chamomile or cumin NE in reinforced the trend
observed with the short-term toxicity bioassay reported
above. Mortality was increased with increasing concentration
and length of exposure for both NEs, but the cumin NE
exhibited higher toxicity with a steep increase in mortality at
concentrations above 50 mg/mL and reaching mortality levels
above 50% after 10 days exposure, unlike chamomile, which
did not reach even 25% mortality at the highest concentration
and longest exposure (Fig. 1).
Hashem, A. S. and Marwa M. Ramadan
Fig. 1. Filled contour maps exhibiting the effects of concentration and exposure time on adult mortality of red flour
beetles provided with cracked wheat treated with essential oil nanoemulsions of chamomile and cumin. The
maps were plotted using regression models, as indicated in the figures, where Z is mortality, X is the essential oil
concentration, and y is the exposure time.
Insect weight gain, food consumption, and feeding
deterrence
The insects surviving were followed for eight days,
after which their gain in weight (i.e., body mass) was
determined, as was the food consumption during the period.
The weight gain of adult insects decreased with increasing the
essential oil concentration with the cumin NE exhibited
higher effect in compromising insect weight gain (Fig. 2A).
A similar trend was observed for food consumption, which
also declined with essential oil concentration and again with
cumin exhibited more drastic effect in compromising the
relative rate of food consumption than chamomile (Fig. 2B).
Fig. 2. Insect weight grain (± SE) (A) and food
consumption (± SE) (B) of red flour beetles
exposed to increasing concentrations of essential
oil nanoemulsions of chamomile and cumin. The
symbols represent the mean of three
independent replicates.
The efficiency of food conversion was estimated
based on the insect weight gain and food consumption with
both essential oils compromised food conversion at
concentrations as low as 25 mg/mL. The rate of such decline
with increasing concentration was similar for both oils, but
cumin imparted slightly higher effect than chamomile (Fig.
3A). In addition, both essential oils also deterred feeding
among flour beetles with a similar rate, as indicated by the
similar slopes of the curves of feeding deterrence with
concentration (Fig. 3B). Feeding deterrence increased with
concentration and the effect of cumin was consistently
stronger than that of chamomile (Fig. 3B).
Fig. 3. Food conversion efficiency (± SE) (A) and feeding
deterrence (± SE) (B) of red flour beetles exposed
to increasing concentrations of essential oil
nanoemulsions of chamomile and cumin. The
symbols represent the mean of three independent
replicates.
Progeny production and grain loss
The insects surviving with were able to reproduce
while maintaining their feeding activity. Although, the
progeny production of the exposed adults of the red flour
beetles decreased with the concentrations of both essential
oils with similar rate, the effect of the cumin NE was always
stronger leading to lower progeny emergence (Fig. 4A). The
J. of Plant Protection and Pathology, Mansoura Univ., Vol 12 (1), January, 2021
cost of the feeding activity and progeny production was the
decrease in grain weight with essential oil concentration (Fig.
4B). The rate of decrease was similar for both essential oils,
but again cumin led to higher declines in grain weight loss
with increasing in concentration than chamomile (Fig. 4B).
Fig. 4. Progeny production (± SE) (A) and grain loss (±
SE) (B) by red flour beetles exposed to increasing
concentrations of essential oil nanoemulsions of
chamomile and cumin. The symbols represent the
mean of three independent replicates.
Discussion
The development of nanoemulsion formulations form
the essential oils of chamomile and cumin was the focus of
the present study aiming their potential use as alternative
insecticides against the red flour beetle. Indeed, we were able
to obtain both nanoemulsions, which favors the use of
essential oils as insecticides minimizing their variability while
improving the physical stability of their bioactive compounds,
protecting them from the interactions with food ingredients.
Because of the subcellular size of the nanoformulations, their
bioactivity is increased through the activation of passive
mechanisms of cell absorption (Berne and Pecora, 2000).
The physicochemical properties of the nanoemulsions
are mainly determined by their zeta potential (mV), poly-
dispersivity (PDI), Z-average (nm), and other related
characteristics (Lett 2016). Nanopesticides, including
nanoemulsions, have a typical particle size range of 50-200
nm (Tadros et al., 2004). Emulsifiers may act as a mechanical
barrier and by forming a surface potential (zeta potential),
which can produce repulsive electrical forces among
approaching oil droplets thus hindering coalescence (Bordes
et al., 2009). High zeta potentials were observed for
nanoemulsions of chamomile and cumin EO (-3.2 and -10.1
mv, respectively), which results from both oil and surfactant
compositions. Zeta Potential values greater than +25 mV or
less than -25 mV typically have high degrees of stability, as
the case with chamomile and cumin EA. Scatterings with a
low zeta potential value will eventually aggregate due to Van
Der Waal inter-particle attractions (Shi et al., 2017). Both of
these effects lead to a narrow range of sample concentrations
that will yield a satisfactory quality result (Hinds, 2012). In
addition, a highly conductive sample (> 5mS/cm) can lead to
electrode polarization and degradation (Patakangas, 2014).
Other studies revealed that particle sizes of nanoemulsions of
essential oils produced by high pressure homogenization and
spontaneous emulsification were similar to those obtained in
this study, as mentioned by Dias et al. (2014).
Poly-dispersity (PDI) is another important property of
nanoformulations. The measurement refers to the uniformity
of droplet size withinthe formulation (Flores et al., 2011).
Therefore, values of PDI lower than 0.2 indicate homogenous
droplet populations, while a 0.3 value represents
heterogeneity (Hoeller et al., 2009). Our results demonstrated
that the nanoemulsion of chamomile was particularly uniform
compared to the nanoemulsion of cumin. Thus, added
homogeneity can be achieved by increasing the viscosity of
the continuous phase of the formulation preparation.
However, this delays instability resulting in oil droplets of
more homogeneous particle size (Arancibia et al., 2016),
which can be further improved for the cumin oil. Regardless
of whether the insecticidal effects of cumin are higher or less
effective compared to chamomile
The search for bio-rational insecticides of natural
origin is on the increase particularly for organic production
systems, because they also consider as a means to minimize
adverse effects of conventional pesticides (Aktar et al. 2009).
In such context, preparation of nanoemulsions has emerged
as a promising alternative to improve EO performance against
arthropod pest species (Damalas and Koutroubas 2018).
Nanoemulsions have received a great deal of attention from
the pharmaceutical sector, for example as potential vehicles
for transdermal delivery of hydrophobic drugs (Shakeel et al.,
2012). Recent advances in agriculture research have also
triggered great interest in the exploration of nanotechnology
(Khot et al. 2012). The objective is usually to increase the
physical stability of the essential oil bioactive compounds.
Nanoemulsions of pesticidal active ingredients have often
been suggested to increase the insecticide uptake, but
supporting data in plant-protection products remains scarce.
However, two recent studies support the hypothesis of
enhanced uptake (Oberdörster et al., 2005). In the first of
these studies, experiments on a series of nanoemulsions of
neem oil showed that the LC50 decreased with droplet size,
which was interpreted as indicating an increased uptake of
smaller droplets. (Anjali et al., 2012).
The bioactivity and persistence of nanoemulsions of
the two tested oils against the red flour beetle exhibited
insecticidal activity, including lethal and sublethal effects.
Increased mortality with nanoemulsions of natural
insecticides were also observed in other studies with stored
product insects (e.g., Nenaah et al. 2015; Oliveira et al. 2017),
and mosquitoes (Oliveria et al., 2016). Most reports followed
the same approach in controlling insect pests, but used
different surfactants, like Tween (Montefuscoli et al., 2014)
and polyethylene glycol (González et al., 2014), and β-
cyclodextrin (Galvão et al., 2015), poly-β-hydroxybutyrate
and poly-ε caprolactone (Carvalho et al., 2015), as
encapsulating agents.
The main point is that the developed nanoemulsion
allowed for the insecticidal use of chamomile and cumin EO.
The concentration-mortality toxicity bioassays performed
indicated that the acute activity of these compounds, which
are not particularly high, but cumin EO seems promising for
further development. Eventual fractioning of use extracts with
higher concentration of main components will likely improve
Hashem, A. S. and Marwa M. Ramadan
the short-term performance of this EO. However, insecticidal
activity goes beyond short-term mortality (Guedes and Cutler,
2014; Guedes et al., 2016, 2017), and in our study we also
assessed a range of sublethal effects of the EO nonemulsion
exposure. Both essential oils and specially cumin essential oil
compromised food consumption, food conversion, insect
development and reproduction, minimizing grain loss. The
effects were always higher for cumin EO reinforcing the
potential of nanoemulsions of cumin EO for further
development aiming stored product protection, wich might
deserve further investigations.
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(EO)
(Matricaria chamomilla L.) (Cuminum cyminum L.) Tribolium castaneum (Hersbt).
(PDI) (cP) (mV) (mS / cm)