Available via license: CC BY 4.0
Content may be subject to copyright.
Plant Science Today, ISSN 2348-1900 (online)
OPEN ACCESS
ARTICLE HISTORY
Received: 14 February 2024
Accepted: 13 March 2024
Available online
Version 1.0 : 31 March 2024
Additional information
Peer review: Publisher thanks Sectional Editor
and the other anonymous reviewers for their
contribution to the peer review of this work.
Reprints & permissions information is avail-
able at https://horizonepublishing.com/
journals/index.php/PST/open_access_policy
Publisher’s Note: Horizon e-Publishing
Group remains neutral with regard to jurisdic-
tional claims in published maps and institu-
tional ailiations.
Indexing: Plant Science Today, published by
Horizon e-Publishing Group, is covered by
Scopus, Web of Science, BIOSIS Previews,
Clarivate Analytics, NAAS, UGC Care, etc
See https://horizonepublishing.com/journals/
index.php/PST/indexing_abstracting
Copyright: © The Author(s). This is an open-
access article distributed under the terms of
the Creative Commons Attribution License,
which permits unrestricted use, distribution
and reproduction in any medium, provided
the original author and source are credited
(https://creativecommons.org/licenses/
by/4.0/)
CITE THIS ARTICLE
Mishra PP, Mishra PR, Adak T, Gowda BG,
Pandi GPG, Golive P, Rath PC, Das SK, Patil
NB. Vitex negundo L. oil nanoemulsion for the
ecofriendly management of Sitophilus oryzae
(L.) and Tribolium castaneum (Herbst) in
stored rice . Plant Science Today (Early Ac-
cess). https://doi.org/10.14719/pst.3391
Abstract
The widespread use of synthetic chemicals as storage protectants makes
food hazardous, endangers human health and develops insect resistance.
Hence, in the present study Vitex negundo L. oil nanoemulsion (VNO NE) was
prepared to manage stored grain pests. V. negundo oil (VNO) had major
compounds like Aromandendrene, β-caryophyllene, Squalene, 3-octen-
5-yne,2,7-dimethyl-, (E)-, 5-(1-isopropenyl-4,5-dimethylbicyclo[4.3.0]nonan-
5-yl)-3-methyl-2-pentenol acetate, Farnesyl bromide, 4-terpeneol and
Elemol. A high-speed homogenizer was used to formulate nanoemulsions of
VNO and studies on their physico-chemical and thermal stability revealed
that, the optimum nanoemulsion had 5% VNO mixed at a 1:2 (w/w) ratio
with tween 80 surfactant. The hydrodynamic diameter, polydispersity index
and mean zeta potential of the nanoemulsion were 166.62 nm, 0.263 and
-3.4 mV respectively and droplet sizes varied from 50 to 200 nm in transmis-
sion electron microscopy. Lethal dose 50 (LD50) values for contact toxicity of
VNO nanoemulsion (VNO NE) were 0.755 and 3.131 μL cm-2 against
Sitophilus oryzae and Tribolium castaneum respectively which were 41.60
and 29.88% less compared to VNO. In case of fumigant toxicity, LD50 value of
VNO NE was 322.28 μL L-1 against S. oryzae which was 26% less than that of
crude oil. Highest repellency increased by 33.33 and 36.58% when treated
with VNO NE in S. oryzae and T. castaneum respectively. Also significant Glu-
tathione S-transferase enzyme inhibition activities observed in VNO NE
treated insects as compared to VNO and control. Thus, VNO NE having
improved eicacy and targeted delivery could contribute towards eco-
friendly sustainable stored grain pest management in rice.
Keywords
Vitex negundo L. oil; GC-MS; nanoemulsion; fumigant toxicity; contact toxicity;
glutathione S-transferase activity
Introduction
Storage is essential for reducing wastage and maintaining food security.
Post-harvest storage loss caused by stored grain pests are a global issue
since they aect both grain quality and quantity. Insect damage accounts
for 2-4.2% of total storage loss (1) in India, it causes a direct and indirect
loss close to Rs. 1300 crores annually (2). More than 600 species of
coleopteran beetles, cause storage loss by damaging and contaminating the
PLANT SCIENCE TODAY
ISSN 2348-1900 (online)
Vol x(x): xx–xx
https://doi.org/10.14719/pst.3391
HORIZON
e-Publishing Group
Vitex negundo L. oil nanoemulsion for the ecofriendly
management of Sitophilus oryzae (L.) and Tribolium castaneum
(Herbst) in stored rice
Prajna Prakash Mishra1, P R Mishra1, Totan Adak2, Basana Gowda G2, Guru Pirasanna Pandi G2, Prasanthi Golive2,
P C Rath2, Susanta Kumar Das3 & Naveenkumar B. Patil2*
1 Department of Entomology, College of Agriculture, OUAT- Bhubaneswar - 751003, India
2 Crop Protection Division, ICAR-National Rice Research Institute, Cuttack - 753006, India
3Department of FRM, College of Forestry, OUAT- Bhubaneswar - 751003, India
*Email: patil2850@gmail.com
RESEARCH ARTICLE
MISHRA ET AL 2
https://plantsciencetoday.online
goods (3).
Rice, consumed by 1.6 billion people throughout the world
as a primary food source of calories and proteins (4).
Additionally, it is rich in carbohydrates, micro-nutrients
and vitamins that are required for growth and nutrition of
human being. Rice is either boiled or crushed into flour.
A variety of by products like breakfast cereals, noodles and
soups can be made from rice. Rice suers enormous losses
due to cosmopolitan stored grain pests like Sitophilus ory-
zae (L.) and Tribolium castaneum (Herbst). According to
United Nations (UN) studies, S. oryzae (L.) and T. castane-
um (Herbst) are the 2 foremost stored grain pests of rice
globally having 90% damage potential within 5-6 months
of infection (5). The Sitophilus oryzae bores a hole in the
kernel and deposits her egg therein and reducing kernels
to mere powder (6). The red flour beetle, T. castaneum
Herbst (Coleoptera: Tenebrionidae) is one of the major
secondary pests of stored commodities and is more de-
structive due to high reproduction rate (7). Insect feeding
not only lessens grain weight, nutritional value and germi-
nability but also contaminates grains, induces odor (due to
noxious secretions, exuviae and feces from storage pests)
that aects the grain quality and mass market by making
it unsuitable for human consumption (8, 9).
From several decades, synthetic pesticides and
fumigants like methyl bromide and phosphine has been
used enormously to control storage pests (10). Repeated
use of these chemicals, make the food toxic due to a rise in
their eective concentration and the environment unsafe
for humans and non-target organisms due to their hazard-
ous residues and slow deterioration rate. Also pests began
to develop resistance to these frequently used chemicals.
In India and Australia, pests develop high resistance
towards phosphine in many occasions that cause failure in
pest control (11). Due to all these drawbacks, entomolo-
gists are shiing their focus to botanicals with a special
emphasis on essential oils as grain protectants. Botanicals
in form of phyto-products such as plant parts (leaf, seed,
bark and root), aqueous or solvent extracts, powders and
volatile oils can be used as novel and more pleasant
replacements for synthetic pesticides (12). They exhibit
toxicity towards large number of storage insects with
minimum chance of developing resistance in pests (13).
Essential oils from plant families like Acoraceae,
Asteraceae, Apiaceae, Lamiaceae, Myrtaceae, Lauraceae
and Rutaceae are aromatic and highly volatile present in
dierent parts like leaves, rhizomes, fruits and bark of
plant those have lethal eect on pests (14). Secondary me-
tabolites like terpenoids, phenolics and alkaloids found in
these oils contain toxicant, fumigant, antifeedant,
repellant and oviposition deterrent properties that
interfere with pests' biochemical, physiological and meta-
bolic processes (15).
More than 2000 species of plants contain phyto-
chemicals having insecticidal properties against storage
pests (16). Nirgundi (Vitex negundo L.), a member of the
Verbenaceae family, is a deciduous shrub that grows
across India in wastelands, mixed open forests at an eleva-
tions of up to 1500 metres. It has 4-10 cm long, smooth,
petiolate, exstipulate leaves with aromatic properties (17).
Essential oil from V. negundo contains bioactive
compounds like iridoids, iridoid glycosides, lignans,
flavonoids, flavones glycosides, sterols, polyphenols and
terpenoids, those are crucial for pest management (18).
V. negundo L. leaf extracts show insecticidal properties
against S. granaries and T. castaneum (19, 20). Despite
meeting several requirements to be an eicient weapon,
essential oil-based pesticides have a number of limita-
tions, including water insolubility, quick degradation,
susceptibility to flocculation, creaming and phase separa-
tion owing to Oswald ripening (21).
An emerging method to address these limitations is
nanoemulsion formulation, where the droplet size ranged
between 0.1 to 200 nm and droplets typically do not
coalesce (22). Oil that has been nano-formulated has
greater physical stability since, it degrades and evaporates
much lesser than its normal form (23). Nano ranged
particle size possesses more surface area and mobility that
penetrate the insect cuticle more eectively and shows
higher insecticidal activity (24). Nanoemulsions have
qualities like water solubility, stability and uniform
dispersion those helps in eective pest management (25).
The active components of nanoemulsion spread and pene-
trate well in target site due to small size (26). Mostly it
eliminates the requirement of high concentrations in
toxicity assessments and the annoyance of side eects on
organisms other than the target pests. For the preparation
of nanoemulsions with a low polydispersity index and
small droplet size, some techniques include high-shear
blending, high-pressure homogenization and ultrasoni-
cation (27). In this study high speed homogenization tech-
nique was used to develop nanoemulsion of VNO which
further characterized and tested for its insecticidal activity
against major stored grain pests of rice viz. S. oryzae (rice
weevil) and T. castaneum (rust red flour beetle).
Materials and Methods
Plant material
The Vitex negundo L. leaves were collected from
Kanheipur village, Cuttack, Odisha, India (Fig. 1a). The
Grain Entomology Laboratory of the Crop Protection
Division, ICAR-National Rice Research Institute, Cuttack,
Odisha (20°45' N latitude, 85°93′ E longitude and 36 m
altitude) maintains a specimen copy. ICAR- NRRI comes
under east and south coastal plains agroclimatic zone of
odisha.
Chemicals
Polyoxyethylene sorbitan monooleate, oen known as
Tween 80, was bought from Merck, India, used as
surfactant in nanoformulation preparation. Glutathione
S-transferase (GSTs) kit was purchased from
Sigma-Aldrich, Merck, India, used for GSTs assay.
Oil extraction
Hydro-distillation method was used for oil extraction in
which dried and chopped leaves were boiled at 70οC for 4 h
in Clevenger apparatus (Fig. 1). Due to the hot water and
3
Plant Science Today, ISSN 2348-1900 (online)
steam, essential oils escape from the oil glands of leaves.
The vapour mixture of water and oil moved to cooled
condenser from which oil was collected. The quantity (in g)
of essential oil obtained at the end of hydro-distillation
was measured. The recovery of oil was presented in %:
Wt. of the oil (g)
Recovery of oil in percentage (%) = X 100
Wt. of sample (g)
(28)
Chemical characterization of Vitex negundo oil
Chemical profiling of the extracted oil was done through
GC–MS/MS equipment (Shimadzu TQ8040, Shimadzu
Corporation, Kyoto, Japan). The oil was diluted 100 times
into hexane before injecting. The capillary column Rxi-5-Sil
MS (30 m×0.25 mm, 0.25 µm) was used in the gas chroma-
tography system. The injector’s temperature was main-
tained at 250 ºC. The temperature of the GC oven was
maintained at 50 ºC for 2 min, then raised to 200 ºC at
5 ºC min-1 and maintained there for 2 min, then raised to
250 ºC at 10 ºC min-1 and maintained there for 2 min and
finally raised to 280 ºC at 15 ºC min-1 and maintained for
5 min. Helium with purity more than 99.99% was utilized
as the carrier gas at continuous flow rate of 1.69 mL min-1
in the split mode with a split ratio of 25 and purge flow of
3.0 mL min-1. The GC run time was 48.0 min. The ion source
temperature was fixed at 200 ºC and interface temperature
was fixed at 250 ºC, the MS was run in scan mode from m/z
40 to 500. Each chemical compound was identified by
comparing the retention indices of the compounds to
C8-C40 alkane standards and by comparing the MS spectra
to the reference spectra in the National Institute of
Standards and Technology (NIST) library (29).
Insect culture
Sitophilus oryzae (rice weevil) and Tribolium castaneum
(rust red flour beetle) were obtained from Grain Ento-
mology Laboratory of Crop Protection Division, National
Rice Research Institute. Culture and maintenance of test
insects were done under laboratory condition at 65 ± 5%
RH (relative humidity) and 28 ± 2 °C throughout the study
period. Rice was the feeding material for S. oryzae
whereas, rice flour was used for T. castaneum. Newly
emerged adults of same age insects of 7-10 days old were
utilized in our experiment. For contact and fumigant
toxicity, 10 numbers of insects per replication and for
repellency, 20 numbers of insects per replication taken.
Preparation of nanoemulsions
Vitex negundo L. oil nano emulsion (VNO NE) was pre-
pared by using Tween 80 as a surfactant, chosen based
on the hydrophilic lipophilic balance of the VNO. A range
of VNO concentrations (2.5, 5 and 7.5%) were taken to
manufa-cture various VNO NEs. VNO and surfactants
were mixed at different ratios on weight basis starting
from 1:1 till 1:3 (w/w). The necessary amount of water
was added to the mixture and it was vortexed (Maxi Mix
II, Thermolyne, USA) for 2-3 min. The bulk emulsion was
homogenized at 3 distinct durations (10, 15 and 20 min)
and 3 different rotation speeds (10000, 15000 and 20000
revolutions per min (rpm)) using a high -speed homoge-
nizer (IKA T25 digital ULTRA TURRAX, T 25 D S22, Germa-
a. V. Negundo leaves b. Chopped leaves
c. Essenal oil collected in condenser d. Hydro-disllaon by Clevenger apparatus
Fig. 1. Oil extraction from V. negundo by hydro-distillation method.
MISHRA ET AL 4
https://plantsciencetoday.online
ny).
Characterization of nanoemulsions
Stability study
To obtain stable VNO NE, stress tests (heating, cooling and
freezing, thawing) were conducted to determine the
stability of emulsion under stress (30, 31). To check phase
separation, prepared nanoemulsions were centrifuged for
30 min at 3000 rpm and at 25 °C. An alternate heating and
cooling stress test cycle was performed at temperatures of
40 °C and 4 °C, with each temperature being altered aer
48 h. Alternate freezing and thawing stress procedures was
conducted at -21 °C and 25 °C for 48 h. Both procedure was
performed in triplicate and repeated twice. The VNO NEs,
those succeed the stress tests were characterized further.
Dynamic Light Scattering (DLS)
Particle size analyzer (Malvern Instruments Pvt Ltd, UK)
was used to determine the hydrodynamic diameter, poly-
dispersity index (PDI) and zeta potential (ζ-potential) of
VNO NEs.
Transmission Electron Microscopy (TEM)
Visualization of morphology and structure of VNO NEs
were carried out using transmission electron microscopy
(TEM). A drop of the nanoemulsion was kept on a carbon-
coated copper grid of size 200 mesh. The grid was dried for
5 min at normal temperature followed by Infra-Red (IR)
lamp drying of 2 h. Micrographs were obtained by the help
of a transmission electron microscope (JEM 2100+, JEOL)
working at 200 kV.
Bio-eicacy test
Contact toxicity
Contact toxicity of VNO against S. oryzae and T. castaneum
was conducted (32) (Fig. 2a). Completely randomized
design (CRD) was followed to conduct this experiment.
Cemented petri-plates with a surface area of 64 cm2 were
used for conducting contact toxicity assay. Bio-eicacy
test of individual oil was done at varying oil doses. Doses
of VNO for dierent treatments against S. oryzae were 0.2,
0.4, 0.6, 0.8, 1.0, 1.5 μL cm-2 and control and for T.
castaneum, doses were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 μL cm-2
and control. For smooth application in cemented petri-
plates, dierent doses of VNO were diluted in soybean oil
to a predetermined amount of 500 μL. In each petriplate
ten adults were released along with 1 g of rice or broken
rice as food. Petri plates were tightly covered with lids
followed by sealing with the help of parain film to avoid
escape of insects and pin holes were created for ventila-
tion. Each treatment was replicated thrice and mortality
was recorded at 24 and 48 h aer treatment (HAT). Lethal
doses (LD) were obtained using 1.5 EPA Probit Analysis
Program soware, where doses of treatments, total
number of insects (30 per treatment) and numbers of dead
insects per treatment were inputted in the probit analysis.
Fumigant toxicity
Fumigation chambers (volume 1150 mL) were used to
conduct fumigant toxicity (33) (Fig. 2b). Ten adults belong-
ing to each insect group were kept in perforated pouch
with 1 g rice, tied with rubber bands and placed inside the
fumigation chamber. Filter paper (Whatman No. 1) was cut
into 64 cm2 pieces and was treated with dierent doses of
VNO (300, 400, 500 and 600 µL per litre air (µL L-1) and
control). Filter paper was placed at the top of the air tight
fumigation chamber near the lid. Each treatment was
replicated thrice and adult mortality was recorded 5 days
aer treatment (DAT). LD10, LD50 and LD90 values were
obtained.
Repellancy study
Glass petri-plate of 64 cm2 consists of a filter paper
(Whatman No. 1, cut into 64 cm2 piece) was used for
repellency study (34). Dierent doses of VNO (1.5, 4.5 and
7.5 µL) were mixed with soybean oil to prepare VNO treat-
ments and volume was made up to 150 µL. Filter paper
was divided in to 2 equal halves in which 1 was treated
with VNO treatment and other half was control i.e treated
with only soybean oil (150 µL). Petri-plates were subjected
to fan drying and insects were released at the middle area
of filter paper. Each treatment was replicated 7 times and
number of insects present in the 2 parts was recorded at 2,
6, 12 HAT. For every considered time interval, the %
repellency of dierent VNO treatment was measured using
the formula:
Nc - Nt
PR (%) = X 100 (35)
Nc + Nt
where, Nc is the number of insects in the untreated half
paper and Nt is the number of insects in the treated half.
Bioeicacy test of VNO NE
Same methods were followed for studying the contact,
fumigant and repellent toxicity of VNO NE as described in
VNO bioeicacy test. Doses of VNO NE were taken based
on LD50 values of contact, fumigant toxicity of VNO. For
contact toxicity against S. oryzae treatment, doses of VNO
NE were 1.05, 1.3, 1.55, 1.8 µL cm-2 and control, against
T. castaneum doses were 4, 4.5, 5, 5.5 µL cm-2 and control.
In case of fumigant toxicity, treatment doses of VNO NE
were 325, 375, 425, 475 µL L-1 and control for S. oryzae.
Lethal doses were calculated in all cases. For repellency
study, treatment of one half filter paper was VNO NEs (150
µL volume of VNO NEs with dierent oil concentrations of
1.5, 4.5 and 7.5 µL) and % repellency was calculated.
Glutathione-S-transferase (GSTs) assay
a b
Fig. 2. Bio eicacy test. (a). Contact toxicity, (b). Fumigant toxicity
5
Plant Science Today, ISSN 2348-1900 (online)
Enzyme extraction
S. oryzae and T. castaneum adults were exposed under
dierent treatments of contact toxicity (LD50 dose of VNO,
LD50 dose of VNO NE and control) at dierent exposure
times (1, 6 and 24 h). Extraction of the enzymes done (36).
Before exposing the adults to bioassay, all the beetles
were pre-weighed and the weight of the insects were
3.62 mg and 6.32 mg (per 3 insects) for S. oryzae and
T. castaneum respectively. Aer the exposure time, both
the treated and untreated samples were kept at -80 °C and
then crushed with 1 mL Dulbecco’s phosphate-buered
saline (DPBS) in eppendorf tube. The crushed sample were
centrifuged at 10000 rpm and the supernetants were
collected in the fresh tube and kept for further analysis.
Enzyme assay
The glutathione S-transferase (GSTs) enzyme activities
were measured by using the commercially available GST
assay kit (CS0410, Sigma- Aldrich, Merck, India). GSTs
activities were recorded in the microplate reader
(EpochTM2 microplate reader, Agilent, United States) at
340 mm and 1-min intervals according to the manufa-
cturer instructions. Change in absorbance was calculated
at 5 min. Total GST activities were calculated from the
CDNB (1-chloro, 2,4-dinitrobenzene) extinction coeicient
(0.0096). The reaction solution had 4 μL of enzyme
solution, 196 μL of substrate solution (192.08 μL of DPBS,
pH 7.2, 200 mM Glutathione reduced and 1.96 μL of
100 mM CDNB (1-chloro-2,4-dinitrobenzene).
Statistical analysis
The Probit regression analysis was done using 1.5 EPA
Probit Analysis Program soware (37). Lethal doses
(LD10, LD50 and LD90) were estimated. GSTs data were
obtained using GEN5 absorbance microplate soware and
analysed using one-way ANOVA at P<0.05 in Microso
excel version 2016.
Results and discussion
Chemical constituents of VNO
Recovery of oil from dried leaves of V. negundo (L.) was
0.65 % which was pale-yellow in colour. According to
earlier reports V. negundo leaves produced 0.4% oil (38)
and 0.5% essential oil by hydro-distillation method (28).
The variation in amount of oil yield might be due to the
genetic make-up of the V. negundo plant population as
well as extraction methods, seasonal variations, environ-
mental, soil and climate conditions (18).
From gas chromatography–mass spectrometry
(GC–MS) technique a total of 40 chemical compounds were
identified constituting almost 70% of oil. Major
compounds of oil were found to be Aromandendrene
(13.21%), β-caryophyllene (7.79%), Squalene (5.48%),
3-octen-5-yne,2,7-dimethyl-,(E)-(5.43%),5-(1-isopropenyl-
4,5-dimethylbicyclo[4.3.0]nonan-5-yl)-3methyl-2-pentenol
acetate (3.59%), Farnesyl bromide (3.51%), 4-terpeneol
(2.97%) and Elemol (2.18%) (Table 1). Many chemical
components from our results such as α-Pinene, β-pinene,
3-octanone, p-cymene, 1,8-cineole, γ -terpinene, Linalool,
4-terpeneol, α-terpineol, β-elemene, β-caryophyllene,
Elemol, Nerolidol, Caryophyllene oxide, epi-α-cadinol,
β-eudesmol corroborated the earlier studies (27, 38, 39).
Also some other compounds Humulane-1,6-dien-3-ol,
Sclareol, Nerolidol, 4-Terpinenol, β-caryophyllene and
β-eudesmol were identified previously which are similar
with our result (28). β- caryophyllene found to be one of
the major compound of V. negundo oil (40). Geographical
and climatic factors can sometimes be the primary causes
of variations in chemical composition, both in terms of
quality and quantity (27).
Preparation and characterization VNO NE
Keeping VNO concentration at 5% with constant ratio of
VNO: surfactant (1:2) several combinations of emulsions
were formulated in order to comprehend the homogeniza-
tion time and homogenizer rotation speed (Table 2).
To achieve the best combining ratio and the maximum
loading capacity of the oil, V. negundo oil and surfactant
were combined in various ratios at a given time (15 min)
and rotation speed (20000 rpm) (Table 3). Varying
combining ratios and oil concentrations led to distinct
colored VNO NE (Fig. 3). Milky white color nanoemulsion
was produced from 2.5% oil concentration and 1:1 (oil,
surfactant ratio) and at ratio 1:2 with same oil concentra-
tion milky VNO NE resulted. Transparent white and super
white coloured emulsions were obtained at 1:2 and 1:1,
VNO and surfactant ratio at 5% oil concentration respec-
tively. At 7.5% VNO concentration, super white emulsion
was observed at a ratio 1:1 of oil to surfactant and turbid
emulsion was formed at a ratio of 1:2.
VNO and surfactant at 1:2 and 1:1 ratio of 5% VNO
concentration were found to be more stable nanoemul-
sions and passed the stress tests as no phase separation
was observed when centrifuged. Phase separation noticed
when loading capacity of VNO was increased to 7.5%
(Table 2). These unstable nanoemulsions may be occurred
due to larger droplets formed by Ostwald ripening and
coalescence of oil (41).
VNO and tween80 at 1:1 and 1:2 ratios of 5% oil con-
centration were further characterized for hydrodynamic
diameter, PDI and ζ-potential (Table 4). Factors like
droplet size, poly dispersity index and zeta potential are
crucial for the stability of nano-formulations as well as
their biological activity (42). The droplet sizes of VNO:
surfactant at 1:1 and 1:2 of 5% oil concentration were
recorded 185.41 and 166.62 nm respectively. This is within
the previously reported range of nanoemulsion droplet
sizes, i.e between 20 to 200 nm (43). Tween 80 sterically
stabilises nanoemulsion droplets due to its high hydro-
philic and lipophilic balancing (44). Smallest droplets
might be produced because of high power energy
generated by the homogenizer (45). An increased stirring
speed led to decrease in particle size (46). Particle size
decreased as a result of the decrease in interfacial free
energy caused by the rise in surfactant concentration,
which could serve as a mechanical barrier to coalescence.
MISHRA ET AL 6
https://plantsciencetoday.online
The attraction forces were weaker and the nanoemulsions
were more stable due to these down sized droplets (47).
Polydispersity index evaluated the non-uniformity
of the formulation's size distribution (48). A PDI value less
than 0.6 showed that the emulsion was more homogenous
and stable (42). VNO: tween 80 at 1:1 and 1:2 of 5% oil
concentration displayed uniformity with low PDI values of
0.281 and 0.263 respectively (Table 4). Homogeneity of
droplet size distribution increased with decreased polydis-
persity value (49). ζ-potential of VNO: tween 80 at 1:1 and
1:2 ratio were -4.3 and -3.4 mV respectively which prevents
quick phase separation. Negative zeta potential caused
more repulsion between droplets thus stabilizes the
Retention time Chemical name Area Area Percentage SI *RI(Li) *RI(Cal)
6.905 β-thujene 19776541 0.18 94 873 930
7.085 α-pinene 40657279 0.37 95 909 937
8.3 3-octen-5-yne, 2,7-dimethyl-, (E)- 590952890 5.43 86 912 979
8.36 β-pinene 52738955 0.48 96 943 981
8.485 Vinyl hexanol 106960214 0.98 93 961 985
8.675 3-octanone 12867508 0.12 95 965 991
8.785 Myrcene 68674820 0.63 95 984 996
8.945 3-octanol 21802761 0.2 95 990 1001
9.515 δ-carene 75026451 0.69 95 1001 1020
9.755 p-cymene 8334998 0.08 94 1008 1029
9.875 D-limonene 68991116 0.63 92 1011 1032
9.955 1,8-cineole 28525768 0.26 96 1016 1035
10.475 β-ocimene 47491778 0.44 96 1026 1053
10.785 γ -terpinene 125885231 1.16 93 1040 1063
11.665 Isoterpinolene 36158415 0.33 96 1082 1093
12.025 Linalool 35519846 0.33 96 1083 1105
12.17 Isopentyl 3-methylbutanoate 16799896 0.15 92 1094 1110
14.335 4-terpeneol 323167077 2.97 91 1161 1185
14.71 α-terpineol 26608713 0.24 95 1179 1197
20.225 β-elemene 20825391 0.19 95 1385 1401
21.025 Aromandendrene 1437590039 13.21 90 1440 1433
21.85 β-caryophyllene 847257487 7.79 90 1449 1466
24.15 Elemol 237257608 2.18 95 1538 1562
24.405 Nerolidol 38813853 0.36 90 1546 1572
24.625 3-hexen-1-ol, benzoate, (Z)- 19649946 0.18 96 1553 1581
24.985 Caryophyllene oxide 84793572 0.78 93 1566 1597
26.09 γ-eudesmol 82972462 0.76 92 1596 1646
26.285 epi-α-cadinol 59907995 0.55 94 1625 1654
26.52 β-eudesmol 54231287 0.5 92 1629 1665
26.585 α-eudesmol 44454785 0.41 89 1635 1668
31.435 Farnesyl bromide 381514220 3.51 80 1764 1899
32.010 Verticillol 42265135 0.39 83 2036 1929
32.815 Cycloeucalenol acetate 272433913 2.5 82 2074 1972
33.26 Humulane-1,6-dien-3-ol 116294413 1.07 86 2080 1996
33.495 Phytol 282736248 2.6 90 2125 2009
33.945 Sclareol 193612163 1.78 91 2228 2037
34.33 5-(1-isopropenyl-4,5-dimethylbicyclo[4.3.0]nonan-
5-yl)-3-methyl-2-pentenol acetate 390435014 3.59 81 2265 2061
34.835 Kolavenol acetate 201479113 1.85 92 2290 2092
35.29 cis-3,14-clerodadien-13-ol 18409197 1.70 93 2411 2126
42.085 Squalene 8172815 5.48 95 2833 2848
Table 1. Chemical composition of Vitex negundo essential oil as identified by gas chromatography coupled with mass spectroscopy.
*SI: Similarity index, RI (Li): Retention index literature, RI (Cal): Retention index calculated.
7
Plant Science Today, ISSN 2348-1900 (online)
formulation (50).
The most crucial method for studying microstruc-
tures is transmission electron microscopy because it di-
rectly generates high-resolution pictures and can record
any coexisting structures and microstructure changes (51).
The droplets of VNO: tween 80 at 1:1 and 1:2 of 5% oil
content were found to be spherical when determined by
transmission electron microscopy (TEM) and particle size
ranged between 100 to 200 nm (Fig. 4). Centre of droplets
were appeared dark, while the surroundings were bright.
Our results from the dynamic light scattering (DLS) tech-
nique's finding of particle size were supported well by data
Rotation
speed (rpm) Time
(Min.) Colour of Original Emulsion Centrifugation Heating and cooling cycle Freezing and thawing
cycle
10,000 15 Transparent white No phase separation Transparent white Transparent white
15,000 15 Transparent white No phase separation Transparent white Transparent white
20,000 15 Transparent white No phase separation Transparent white Transparent white
20,000 10 Transparent white No phase separation Transparent white Transparent white
20,000 20 Transparent white No phase separation Transparent white Transparent white
Table 2. Thermodynamic parameters of nanoemulsion (5% V. negundo oil concentration and V. negundo oil: surfactant :: 1:2) with respect to dierent time and
rotation speed.
*rpm: rotations per minute, Min.: minute.
Oil
Conc. V. negundo oil:
Surfactant Colour of Original
Emulsion Centrifugation Heating and cooling cycle Freezing and thawing
cycle
2.5% 1:1 Milky white No phase separation Milky white Milky white
2.5% 1:2 Milky No phase separation Translucent Translucent
5% 1:1 Super white No phase separation Super white Super white
5% 1:2 Transparent white No phase separation Transparent white Transparent white
5% 1:3 Milky white Phase separation Suspended particle Milky white
7.5% 1:1 Super white Phase separation S uspended particle Suspended particle
7.5% 1:2 Turbid Phase separation Suspended particles Suspended particle
Table 3. Thermodynamic characterization of dierent V. negundo nanoemulsion formulations at fixed time (15 min) and rotation speed (20000 rpm).
Fig. 3. Dierent V. negundo nanoemulsions (VNO NEs) (rotation speed : 20000
rpm, duration : 15 min).
*Nanoemulsion 1 *Nanoemulsion 2
Hydrodynamic diameter (nm) 185.41nm 166.62 nm
Polydispersity index 0.281 0.263
Mean zeta potential (mV) -4.3 mV -3.4 mV
Table 4. Particle size, polydispersity index and zeta potential of Vitex negundo
oil nano-emulsions (VNO NE).
*Nanoemulsion 1: Contain 5% VNO and VNO :surfactant at 1:1 ratio,
*Nanoemulsion 2: Contain 5% VNO and VNO: surfactant at 1:2 ratio
a
Fig. 4. Transmission electron microscopic view of V. negundo oil
nanoemulsions. (a). V. negundo oil nanoemulsion (5%, 1:1), (b) V. negundo
oil nanoemulsion (5%, 1:2).
b
MISHRA ET AL 8
https://plantsciencetoday.online
got from TEM. Particle size from TEM view of lime oil
nanoemulsion had size more or less similar to our results
i.e ranged between 20-200 nm (52). Because of the extraor-
dinary small size of nanoemulsion, Brownian motion may
have been more prominent and gravitational force less
eective, limiting sedimentation or flocculation, which
may have contributed towards its stability (53).
Bioeicacy study
Bioeicacy test conducted against S. oryzae and
T. castaneum and motalities were recorded (Fig. 5).
The eicacy of VNO NE (VNO: tween 80 at 1:2) and bulk oil
were compared. At 24 HAT, the LD50 values for contact tox-
icity of VNO NE (a.i.) against S. oryzae and T. castaneum
were 0.755 and 3.666 μL cm-2 which were 41.60 and 29.88%
less respectively, compared to VNO. However the values of
LD50 values of VNO NE (a.i.) at 48 HAT were 0.704 and 3.480
μL cm-2 against S. oryzae and T. castaneum respectively
which were 18.14 and 17.12% less respectively than crude
oil (Table 5). Results depict that secondary metabolites of
essential oils like terpenoids and sesquiterpenoids were
responsible for contact, fumigant and ingestion toxicity of
essential oil (54). Terpenes, notably monoterpenes, were
assumed to be the principal components responsible for
essential oils' eectiveness against insect pests (55). Thus,
it can be said that several chemicals, such as
β-caryophyllene, Terpinen-4-ol, linalool, α-humulene and
1,8-cineole, may be responsible for contact toxicity to-
wards test insects. An increase in eicacy of formulated
nanoemulsions over eucalyptus oil against S. oryzae and
T. castaneum was observed (56). Superior larvicidal activi-
ties of VNO NE than VNO against Aedes aegypti L. were re-
ported previously (28). Also it was reported that
nanoemulsion of P. anisum showed toxicity against
T. castaneum (57). Nanoparticles had increased surface
area and mobility, allowing them to penetrate the insect
cuticle more eiciently, resulting in higher insecticidal ei-
cacy (24). Due to their small size in comparison to bulk oil,
a
b
Fig. 5. Death of insects due to bio-eicacy. (a). Dead individuals of S.
oryzae, (b). Dead individuals of T. castaneum.
Time
(HAT)
No. of
insects
tested
Lethal dose
50 (LD50)
(a.i.)
(μL/Cm2)
95% fiducial limits Lethal dose
10 (LD10)
(a.i.)
(μL/Cm2)
Lethal dose
90 (LD90)
(a.i.)
(μL/Cm2)
Slope Standard
error
Χ2
calculated df P value
Lower Upper
*Bulk VNO
Sitophilus oryzae (Rice weevil)
24 180 1.327 0.717 2.455 0.124 14.181 1.248 0.136 0.997 4 0.910251
48 180 0.860 0.420 1.763 0.050 14.833 1.037 0.159 0.998 4 0.910099
Tribolium castaneum (Red flour beetle)
24 180 5.228 3.014 9.067 0.753 36.309 1.525 0.122 0.994 4 0.910704
48 180 4.199 2.383 7.398 0.518 34.025 1.410 0.125 0.996 4 0.910402
*VNO NE
Sitophilus oryzae (Rice weevil)
24 180 0.775 0.606 0.992 0.425 1.415 4.890 0.055 0.995 2 0.608049
48 180 0.704 0.538 0.921 0.388 1.280 4.997 0.059 0.918 2 0.631915
Tribolium castaneum (Red flour beetle)
24 180 3.666 3.131 4.319 2.237 6.010 5.988 0.036 0.996 2 0.607745
48 180 3.480 2.953 4.101 2.10 5.581 6.246 0.036 0.994 2 0.608353
Table 5. Contact toxicity of Vitex negundo oil and its nanoemulsion against important stored grain pests of rice.
*Bulk VNO: Vitex negundo oil, VNO NE: Nanoemulsion of Vitex negundo oil (5% VNO; VNO and surfactant were mixed at 1:2 ratio), HAT: Hours aer treatment
9
Plant Science Today, ISSN 2348-1900 (online)
active components in nanoemulsions distribute and pene-
trate well in the target site (26).
In terms of fumigant toxicity, the LD50 value of VNO
NE (a.i.) for S. oryzae was 322.28, µL L-1, which was 26% less
than that of crude oil (Table 6). Terpinen-4-ol had major
role in fumigation against stored grain pests (58). Smaller
particle size of nanoemulsion has a significant impact on
pesticide activity by accelerating insecticide penetration
through the insect cuticle (59). In our result T. castaneum
demonstrated resistance to both VNO and VNO NE fumi-
gant toxicity which may be due to it’s strong exoskeleton
(60).
Highest repellency of VNO NE was increased by
33.33 and 30.14% for S. oryzae and T. castaneum respec-
tively than VNO (Table 7). Common monoterpenoids
present in VNO like linalool and terpinen-4-ol had been
proven to have repellent properties (61). The essential oil's
potent repellent properties were connected to the
presence of α-pinene and limonene (50). Our results
demonstrated the higher repellency of nanoemulsions
over bulk oil which is similar to the earlier studies that,
Citrus sinensis (sweet orange) oil nanoemulsion was more
poisonous and repellent than regular oil against targeted
pests (34). Our results are in conformity with previous
results, that essential oil nanoemulsions were widely
employed as an eective insect repellent, alternative to
Time
(DAT)
No. of
insects
tested
Lethal
dose 50
(LD50)
(μL/L)
95% fiducial
limits
Lethal
dose 10
(LD10)
((μL/L)
Lethal
dose 90
(LD90) (μL/
L)
Slope Standard
error
χ2
calculated df P value
Lower Upper
*Bulk VNO
Sitophilus oryzae (Rice weevil)
5 120 435.55 317.67 597 156.69 1210.69 2.888 0.070 0.983 2 0.611708
*VNO NE
Sitophilus oryzae (Rice weevil)
5 120 322.28 284.79 364.71 224.75 462.14 8.253 0.027 0.988 2 0.610181
Table 6. Fumigant toxicity of Vitex negundo oil and its nanoemulsion against important stored grain pests of rice
*Bulk VNO: Vitex negundo oil, VNO NE: Nanoemulsion of Vitex negundo oil (5% VNO; VNO and surfacta nt were mixed at 1:2 ratio), DAT: Days aer treatment
Concentration
(μL/64 Cm2) 2 HAT 6 HAT 12 HAT
*Bulk VNO
Sitophilus oryzae
1.5 30.00 18.57 12.86
4.5 47.14 35.71 25.71
7.5 57.14 45.71 28.57
S.E(m) ± 4.07 4.90 3.56
CD (0.05) 12.09 14.57 10.58
Tribolium castaneum
1.5 20.00 14.29 11.43
4.5 32.86 21.43 17.14
7.5 41.43 27.14 20.00
S.E(m) ± 3.12 3.33 2.86
CD (0.05) 9.28 9.90 8.49
*VNO NE
Sitophilus oryzae
1.5 41.43 32.86 24.29
4.5 60.00 48.57 37.14
7.5 75.71 60.00 48.57
S.E(m) ± 4.59 4.39 4.36
CD (0.05) 13.64 13.04 12.97
Tribolium castaneum
1.5 31.43 24.29 18.57
4.5 42.86 35.71 20.57
7.5 55.71 51.43 40.00
S.E(m) ± 5.35 5.06 3.53
CD (0.05) 15.88 15.04 10.49
Table 7. Percentage repellency of Vitex negundo oil and its nanoemulsion
against important stored grain pests of rice.
*Bulk VNO: Vitex negundo oil, VNO NE: Nanoemulsion of Vitex negundo oil
(5% VNO; VNO and surfactant were mixed at 1:2 ratio), HAT: Hours aer treat-
ment.
a
b
Fig. 6. GSTs inhibition activities of V. negundo oil and its nanoemulsion (VO:
Treated with Vitex negundo oil; VN: Treated with Vitex negundo oil nanoemul-
sion) in test species. (a). CDNB product/mg/min) in S. oryzae adults, (b). CDNB
product/mg/min) in T. castaneum adults.
MISHRA ET AL 10
https://plantsciencetoday.online
synthetic chemical pesticides (62).
Biochemical assay
The inhibitory activities of GSTs demonstrated that GSTs
levels in nanoemulsion treated beetles were significantly
lower than the treated beetles of bulk oil and untreated
beetles (Fig. 6). Aer exposed to plant extracts, some
detoxifying enzymes in target insects' tissues and organs
were eliminated or inhibited (63). Earlier, it has been
demonstrated that essential oil nanoemulsions prevent
GSTs activity in stored grain pests (64).
Conclusion
Vitex negundo oil contains major compounds like Aro-
mandendrene, β-caryophyllene, Squalene, 3-octen-5-
yne,2,7-dimethyl-, (E)-, Farnesyl bromide, 4-terpeneol and
Elemol which could be responsible for insecticidal proper-
ties of the oil. Optimum VNO NE had 5% bulk oil mixed at a
1:2 (w/w) ratio with surfactant and prepared using a high-
speed homogeniser at 20000 rpm for 15 min. VNO NE was
more eective than VNO against Sitophilus oryzae and Tri-
bolium castaneum in terms of insecticidal activities as it
has enhanced contact toxicity, fumigant toxicity and
repellency. Significant reduction in GSTs activities ob-
served in VNO NE treated insects as compared to control.
In future nanoformulation of essential oil will be emerged
as a novel alternative to synthetic pesticides which could
protect stored grains from pests more eiciently and with
low doses.
Acknowledgements
The authors are thankful to the Director, ICAR-National
Rice Research Institute, Cuttack for the financial and
logistic support.
Authors’ contributions
NB, PR and TA conceptualised and supervised the research
design and experimental planning. PP carried out the
experiment and analysis. GP participated in the GSTs
enzyme assay. BG, PG, PC and SD participated in the statis-
tical analysis. All authors read and approved the final
manuscript.
Compliance with ethical standards
Conflict of interest: Authors do not have any conflict of
interests to declare.
Ethical issues: None.
References
1. Kumar D, Kalita P. Reducing postharvest losses during storage
of grain crops to strengthen food security in developing coun-
tries. Foods. 2017;6(1):8. https://doi.org/10.3390/foods6010008
2. Anonymous. Importance of storage of grains. Indian Grain Stor-
age Management and Research Institute; 2019. https://
www.igmri.dfpd.gov.in/igmri/storage
3. Yang FL, Liang GW, Xu YJ, Lu YY, Zeng L. Diatomaceous earth
enhances the toxicity of garlic, Allium sativum, essential oil
against stored-product pests. Journal of Stored Products Re-
search. 2010;46(2):118-23. https://doi.org/10.1016/
j.jspr.2010.01.001
4. Guru-Pirasanna-Pandi G, Adak T, Gowda B, Patil N, Annamalai
M, Jena M. Toxicological eect of underutilized plant,
Cleistanthus collinus leaf extracts against two major stored grain
pests, the rice weevil, Sitophilus oryzae and red flour beetle,
Tribolium castaneum. Ecotoxicology and Environmental Safety.
2018;154:92-99. https://doi.org/10.1016/j.ecoenv.2018.02.024
5. Sallam MN. Insect damage: Damage on post-harvest. In: Mejia D,
Lewis B (Eds.). Compendium on postharvest operations. Inter-
national Centre of Insect Physiology and Ecology (ICIPE), Nairo-
bi, Kenya. 2014;pp.2-25. http://www.fao.org/3/aav013e.pdf
6. Srivastava C, Subramanian S. Storage insect pests and their
damage symptoms: An overview. Indian Journal of Entomology.
2016;78(special):53-58. https://doi.org/10.5958/0974-
8172.2016.00025.0
7. Oppert B, Elpidina EN, Toutges M, Mazumdar-Leighton S. Micro-
array analysis reveals strategies of Tribolium castaneum larvae
to compensate for cysteine and serine protease inhibitors. Com-
parative Biochemistry and Physiology Part D: Genomics and
Proteomics. 2010;5(4):280-87. https://doi.org/10.1016/
j.cbd.2010.08.001
8. Vassanacharoen P, Pattanapo W, Luecke W, Vearasilp S. Control
of Sitophilus oryzae by radio frequency heat treatment as alter-
native phytosanitary processing in milled rice. Conference on
International Agricultural Research for Development. 2008;pp.1-
4.
9. Bekon AK, Fleurat-Lessard F. Assessment of dry matter loss and
frass production in cereal grain due to successive attack by
Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). Interna-
tional Journal of Tropical Insect Science. 1992;13(1):129-36.
https://doi.org/10.1017/S1742758400013989
10. Boyer S, Zhang H, Lempérière G. A review of control methods
and resistance mechanisms in stored-product insects. Bulletin
of Entomological Research. 2012;102(2):213-29. https://
doi.org/10.1017/S0007485311000654
11. Donahaye EJ. Current status of non-residual control methods
against stored product pests. Crop Protection. 2000;19(8-
10):571-76. https://doi.org/10.1016/S02612194(00)00074-0
12. Caballero-Gallardo K, Olivero-Verbel J, Stashenko EE. Repellent
activity of essential oils and some of their individual constitu-
ents against Tribolium castaneum Herbst. Journal of Agricultur-
al and Food chemistry. 2011;59(5):1690-96. https://
doi.org/10.1021/jf103937p
13. Dinesh DS, Kumari S, Kumar V, Das P. The potentiality of botani-
cals and their products as an alternative to chemical insecti-
cides to sandflies (Diptera: Psychodidae): A review. Journal of
Vector Borne Diseases. 2014;51(1):1.
14. Hosseini SF, Zandi M, Rezaei M, Farahmandghavi F. Two-step
method for encapsulation of oregano essential oil in chitosan
nanoparticles: Preparation, characterization and in vitro release
study. Carbohydrate Polymers. 2013;95(1):50-56. https://
doi.org/10.1016/j.carbpol.2013.02.031
15. Tripathi AK, Upadhyay S, Bhuiyan M, Bhattacharya P. A review
on prospects of essential oils as biopesticide in insect-pest man-
agement. J Pharmacogn Phytother. 2009;1:52-63.
16. Rajendran S, Sriranjini V. Plant products as fumigants for stored
-product insect control. Journal of Stored Products Research.
2008;44(2):126-35. https://doi.org/10.1016/j.jspr.2007.08.003
17. Devi G. Medicinal plant: Vitex negundo. International Journal of
Current Research. 2021;13:17592-94. https://doi.org/10.24941/
ijcr.41524.05.2021
18. Padalia RC, Verma RS, Chauhan A, Chanotiya CS, Thul S. Phyto-
11
Plant Science Today, ISSN 2348-1900 (online)
chemical diversity in essential oil of Vitex negundo L. popula-
tions from India. Records of Natural Products. 2016;10(4).
19. Jawalkar N, Zambare S. Bioinsecticidal activity of Vitex negundo
L. (Family: Verbenaceae) leaf extracts against Sitophilus granari-
us L. in stored maize grains. Journal of Entomology and Zoology
Studies. 2020;8(2):1532-38. https://doi.org/10.22271/
j.ento.2020.v8.i2z.6643
20. Chowdhury NY, Islam W, Khalequzzaman M. Insecticidal activity
of compounds from the leaves of Vitex negundo (Verbenaceae)
against Tribolium castaneum (Coleoptera: Tenebrionidae). Inter-
national Journal of Tropical Insect Science. 2011;31(3):174-81.
https://doi.org/10.1017/S1742758411000221.
21. Simonazzi A, Cid AG, Villegas M, Romero AI, Palma SD,
Bermúdez JM. Nanotechnology applications in drug controlled
release. In: Drug targeting and stimuli sensitive drug delivery
systems. William Andrew Publishing. 2018;pp. 81-116. https://
doi.org/10.1016/B978-0-12-813689-8.00003-3
22. Abouelkassem S, Abdelrazeik AB, Rakha OM. Nanoemulsion of
jojoba oil, preparation, characterization and insecticidal activity
against Sitophilus oryzae (Coleoptera: Curculionidae) on wheat.
International Journal of Agriculture Innovations and Research.
2015;4(1):72-75.
23. Martín Á, Varona S, Navarrete A, Cocero MJ. Encapsulation and
co-precipitation processes with supercritical fluids: Applications
with essential oils. The Open Chemical Engineering Journal.
2010;4(1). https://doi.org/10.2174/1874123101004010031
24. Margulis-Goshen K, Magdassi S. Nanotechnology: An advanced
approach to the development of potent insecticides. Advanced
Technologies for Managing Insect Pests. 2013;295-314. https://
doi.org/10.1007/978-94-007-4497-4_15
25. Song S, Liu X, Jiang J, Qian Y, Zhang N, Wu Q. Stability of triazo-
phos in self-nanoemulsifying pesticide delivery system. Colloids
and Surfaces A: Physicochemical and Engineering Aspects.
2009;350(1-3):57-62. https://doi.org/10.1016/
j.colsurfa.2009.08.034
26. Pavoni L, Pavela R, Cespi M, Bonacucina G, Maggi F, Zeni V et al.
Green micro-and nanoemulsions for managing parasites, vec-
tors and pests. Nanomaterials. 2019;9(9):1285. https://
doi.org/10.3390/nano9091285
27. Ghosh V, Mukherjee A, Chandrasekaran N. Formulation and
characterization of plant essential oil based nanoemulsion:
Evaluation of its larvicidal activity against Aedes aegypti. Asian
Journal of Chemistry. 2013;25 (Supplementary Issue):S321.
28. Balasubramani S, Rajendhiran T, Moola AK, Diana RK. Develop-
ment of nanoemulsion from Vitex negundo L. essential oil and
their eicacy of antioxidant, antimicrobial and larvicidal activi-
ties (Aedes aegypti L.). Environmental Science and Pollution
Research. 2017;24:15125-33. https://doi.org/10.1007/s11356-
017-9118-y
29. Anonymous. NIST/EPA/NIH mass spectral library (NIST 17) and
NIST mass spectral search program (Version 2.3).
30. Shafiq S, Shakeel F, Talegaonkar S, Ahmad FJ, Khar RK, Ali M.
Development and bioavailability assessment of ramipril
nanoemulsion formulation. European Journal of Pharmaceutics
and Biopharmaceutics. 2007;66(2):227-43. https://
doi.org/10.1016/j.ejpb.2006.10.014
31. Sugumar S, Clarke SK, Nirmala MJ, Tyagi BK, Mukherjee A, Chan-
drasekaran N. Nanoemulsion of eucalyptus oil and its larvicidal
activity against Culex quinquefasciatus. Bulletin of Entomologi-
cal Research. 2014;104(3):393-402. https://doi.org/10.1017/
S0007485313000710
32. Patil NB, Adak T, Pandi GGP, Gowda GB, Jena M. Ecofriendly
approach for rice weevil (Sitophilus oryzae) (Coleoptera: Curculi-
onidae) management using fumigant oils. In: Proceedings of the
10th International Conference on Controlled Atmosphere and
Fumigation in Stored Products (CAF2016). CAF Permanent Com-
mittee Secretariat, Winninpeg, Canada. 2016;pp. 16-21.
33. Lee BH, Choi WS, Lee SE, Park BS. Fumigant toxicity of essential
oils and their constituent compounds towards the rice weevil,
Sitophilus oryzae (L.). Crop Protection. 2001;20(4):317-20.
https://doi.org/10.1016/S0261-2194(00)00158-7
34. Giunti G, Palermo D, Laudani F, Algeri GM, Campolo O, Palmeri
V. Repellence and acute toxicity of a nano-emulsion of sweet
orange essential oil toward two major stored grain insect pests.
Industrial Crops and Products. 2019;142:111869. https://
doi.org/10.1016/j.indcrop.2019.111869
35. Zhang JS, Zhao NN, Liu QZ, Liu ZL, Du SS, Zhou L, Deng ZW.
Repellent constituents of essential oil of Cymbopogon distans
aerial parts against two stored-product insects. Journal of Agri-
cultural and Food Chemistry. 2011;59(18):9910-15. https://
doi.org/10.1021/jf202266n
36. Bullangpoti V, Wajnberg E, Audant P, Feyereisen R. Antifeedant
activity of Jatropha gossypifolia and Melia azedarach senescent
leaf extracts on Spodoptera frugiperda (Lepidoptera: Noctuidae)
and their potential use as synergists. Pest Management Science.
2012;68(9):1255-64. https://doi.org/10.1002/ps.3291
37. EPA Probit analysis (2012) Version 1.5. http://www.epa.gov/
nerleerd/stat2.htm
38. Issa M, Chandel S, Singh HP, Batish DR, Kohli RK, Yadav SS, Ku-
mari A. Appraisal of phytotoxic, cytotoxic and genotoxic poten-
tial of essential oil of a medicinal plant Vitex negundo. Industrial
Crops and Products. 2020;145:112083. https://doi.org/10.1016/
j.indcrop.2019.112083
39. Suganthi N, Sonal D. Phytochemical constituents and pharma-
cological activities of Vitex negundo Linn. Journal of Chemical
and Pharmaceutical Research. 2016;8(2):800-87.
40. Ghannadi A, Bagherinejad MR, Abedi D, Jalali M, Absalan B,
Sadeghi N. Antibacterial activity and composition of essential
oils from Pelargonium graveolens L'Her and Vitex agnus-castus
L. Iranian Journal of Microbiology. 2012;4(4):171.
41. Shakeel F, Haq N, Al-Dhfyan A, Alanazi FK, Alsarra IA. Chemopre-
vention of skin cancer using low HLB surfactant nanoemulsion
of 5-fluorouracil: A preliminary study. Drug Delivery. 2015;22
(4):573-80. https://doi.org/10.3109/10717544.2013.868557
42. Danaei M, Dehghankhold M, Ataei S, Hasanzadeh Davarani F,
Javanmard R, Dokhani A et al. Impact of particle size and poly-
dispersity index on the clinical applications of lipidic nanocarri-
er systems. Pharmaceutics. 2018;10(2):57. https://
doi.org/10.3390/pharmaceutics10020057
43. Ostertag F, Weiss J, McClements DJ. Low-energy formation of
edible nanoemulsions: Factors influencing droplet size pro-
duced by emulsion phase inversion. Journal of Colloid and In-
terface Science. 2012;388(1):95-102. https://doi.org/10.1016/
j.jcis.2012.07.089
44. Heydari M, Amirjani A, Bagheri M, Sharifian I, Sabahi Q. Eco-
friendly pesticide based on peppermint oil nanoemulsion: Prep-
aration, physicochemical properties and its aphicidal activity
against cotton aphid. Environmental Science and Pollution
Research. 2020;27:6667-79. https://doi.org/10.1007/s11356-019-
07332-y
45. Lee L, Norton IT. Comparing droplet breakup for a high-pressure
valve homogeniser and a Microfluidizer for the potential pro-
duction of food-grade nanoemulsions. Journal of Food Engi-
neering. 2013;114(2):158-63. https://doi.org/10.1016/
j.jfoodeng.2012.08.009
46. Barzegar H, Mehrnia MA, Nasehi B, Alipour M. Fabrication of
peppermint essential oil nanoemulsions by spontaneous meth-
od: Eect of preparing conditions on droplet size. Flavour and
Fragrance Journal. 2018;33(5):351-56. https://doi.org/10.1002/
j.3455
MISHRA ET AL 12
https://plantsciencetoday.online
47. Tadros T, Izquierdo P, Esquena J, Solans C. Formation and sta-
bility of nano-emulsions. Advances in Colloid and Interface Sci-
ence. 2004;108:303-18. https://doi.org/10.1016/j.cis.2003.10.023
48. Martinez NY, Andrade PF, Durán N, Cavalitto S. Development of
double emulsion nanoparticles for the encapsulation of bovine
serum albumin. Colloids and Surfaces B: Biointerfaces.
2017;158:190-96. https://doi.org/10.1016/j.colsurfb.2017.06.033
49. Kotta S, Khan AW, Ansari SH, Sharma RK, Ali J. Formulation of
nanoemulsion: A comparison between phase inversion compo-
sition method and high-pressure homogenization method. Drug
delivery. 2015;22(4):455-66. https://
doi.org/10.3109/10717544.2013.866992
50. Lima TS, Silva MF, Nunes XP, Colombo AV, Oliveira HP, Goto PL
et al. Cineole-containing nanoemulsion: Development, stability
and antibacterial activity. Chemistry and Physics of Lipids.
2021;239:105113. https://doi.org/10.1016/
j.chemphyslip.2021.105113
51. Chang H, Kosari F, Andreadakis G, Alam MA, Vasmatzis G, Bashir
R. DNA-mediated fluctuations in ionic current through silicon
oxide nanopore channels. Nano Letters. 2004;4(8):1551-56.
https://doi.org/10.1021/nl049267c
52. Liew SN, Utra U, Alias AK, Tan TB, Tan CP, Yussof NS. Physical,
morphological and antibacterial properties of lime essential oil
nanoemulsions prepared via spontaneous emulsification meth-
od. LWT. 2020;128:109388. https://doi.org/10.1016/
j.lwt.2020.109388
53. Fernandes CP, de Almeida FB, Silveira AN, Gonzalez MS, Mello
CB, Feder D et al. Development of an insecticidal nanoemulsion
with Manilkara subsericea (Sapotaceae) extract. Journal of
Nanobiotechnology. 2014;12:1-9. https://doi.org/10.1186/1477-
3155-12-22
54. Govindarajan M, Benelli G. Facile biosynthesis of silver nanopar-
ticles using Barleria cristata: Mosquitocidal potential and bio-
toxicity on three non-target aquatic organisms. Parasitology
Research. 2016;115:925-35. https://doi.org/10.1007/s00436-015-
4817-0
55. Rani KS, MM RMM. A review on development of nano formula-
tions of essential oils for stored grain pest management.
56. Adak T, Barik N, Patil NB, Gadratagi BG, Annamalai M, Mukher-
jee AK, Rath PC. Nanoemulsion of eucalyptus oil: An alternative
to synthetic pesticides against two major storage insects
(Sitophilus oryzae (L.) and Tribolium castaneum (Herbst)) of rice.
Industrial Crops and Products. 2020;143:111849. https://
doi.org/10.1016/j.indcrop.2019.111849
57. Hashem AS, Awadalla SS, Zayed GM, Maggi F, Benelli G . Pimpi-
nella anisum essential oil nanoemulsions against Tribolium
castaneum-insecticidal activity and mode of action. Environ-
mental Science and Pollution Research. 2018;25:18802-12.
https://doi.org/10.1007/s11356-018-2068-1
58. Wang Y, Zhang LT, Feng YX, Guo SS, Pang X, Zhang D et al. Insec-
ticidal and repellent eicacy against stored-product insects of
oxygenated monoterpenes and 2-dodecanone of the essential
oil from Zanthoxylum planispinum var. dintanensis. Environmen-
tal Science and Pollution Research. 2019;26:24988-97. https://
doi.org/10.1007/s11356-019-05765-z
59. Kasai S, Komagata O, Itokawa K, Shono T, Ng LC, Kobayashi M,
Tomita T. Mechanisms of pyrethroid resistance in the dengue
mosquito vector, Aedes aegypti: Target site insensitivity, pene-
tration and metabolism. PLoS Neglected Tropical Diseases.
2014;8(6):e2948. https://doi.org/10.1371/journal.pntd.0002948
60. Shamjana U, Grace T. Review of insecticide resistance and its
underlying mechanisms in Tribolium castaneum. In: Insecti-
cides. IntechOpen. 2021. https://doi.org/10.5772/
intechopen.100050
61. Kamanula JF, Belmain SR, Hall DR, Farman DI, Goyder DJ,
Mvumi BM et al. Chemical variation and insecticidal activity of
Lippia javanica (Burm. f.) Spreng essential oil against Sitophilus
zeamais Motschulsky. Industrial Crops and Products.
2017;110:75-82. https://doi.org/10.1016/j.indcrop.2017.06.036
62. Nethaji DK, Parambil KA. Development and applications of nano
emulsion in food technology. International Journal of Science,
Engineering and Management. 2017;2(12):60-61.
63. Ramsey JS, Rider DS, Walsh TK, De Vos M, Gordon KH, Ponnala L
et al. Comparative analysis of detoxification enzymes in
