Article

pubs.acs.org/JAFC

Assessment of Toxicity, Antifeedant Activity, and Biochemical

Responses in Stored-Grain Insects Exposed to Lethal and Sublethal
Doses of Gaultheria procumbens L. Essential Oil
Kiran S and Bhanu Prakash*
Department of Food Protectants and Infestation Control, CSIRCentral Food Technological Research Institute, Mysore 570020,
India

ABSTRACT: The present study was undertaken to investigate the insecticidal activity of chemically characterized Gaultheria
procumbens essential oil (EO) and its mode of action against the Coleopteran insects Sitophilus oryzae and Rhyzopertha dominica.
Gas chromatographymass spectrometry results depicted methyl salicylate (MS) as the major compound (96.61%) of EO. EO
and its major compound methyl salicylate (MS) showed 100% mortality at 150 and 5.0 L/L air against S. oryzae and R.
dominica, respectively, on 24 h of exposure. The in vivo percent inhibition of AChE activity ranged between 6.12 and 27.50%. In
addition, changes in the antioxidative defense system, superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH),
and oxidized glutathione (GSSG), in test insects were estimated. A signicant dose-dependent response in all test parameters was
observed. The results demonstrated that G. procumbens EO could play a signicant role in the formulation of EO-based
insecticides for the management of stored-grain insects.
KEYWORDS: acetylcholinesterase, antioxidant defense system, essential oil, plant-based insecticide, Gaultheria procumbens

INTRODUCTION
Sitophilus oryzae L. (Coleoptera: Curculionidae) and Rhyzoper-
tha dominica F. (Coleoptera: Bostrychidae) are the two
foremost insect pest species of food grainswheat, barley,
rye, oats, and their nished products. Each of their stages, such
as eggs, larvae, and pupae, feed and develop concealed within
the seed kernels, causing huge losses to the aected
commodities.1 Several synthetic insecticides such as phosphine,
chloropyrifos, malathion, and pyrethroids are currently used to
prevent insect pest infestation.2 However, some negative
consequences, such as the development of resistance in treated
pests, residual toxicity, and environmental problems associated
with the use of the synthetic insecticides, have led to the search
for eective biorational and eco-friendly insecticides.2,3
In this prospect, essential oils (EOs) often have strong
fumigant action, low mammalian toxicity, reduced eect on
antileishmanial, antioxidant, and antidiabetic agent has been
well explored in the literature.1416 G. procumbens essential oil
has been reported for its insecticidal and antifeedant activities
against a wide range insect pests.17,18 However, the literature is
silent on the in vivo ecacy of G. procumbens EO on AChE
activity and antioxidative defense systems such as superoxide
dismutase (SOD), catalase (CAT), reduced glutathione (GSH),
and oxidized glutathione (GSSG) in S. oryzae and R. dominica.
The present study explores the chemical prole of G.
procumbens EO and its ecacy against the insect pest S. oryzae
and R. dominica. In addition, eects of lethal and sublethal
doses of EO and its major compound methyl salicylate (MS) to
the insect pest mortality and feeding deterrence have been
studied. We further investigated the possible mode of action of
test EO and MS in terms of their eect on AChE enzyme
activity and antioxidative defense system (SOD, CAT, and
GSH/GSSH).

nontarget organism, and less/no persistence activity, hence,
could be considered as potential biorational alternatives to
synthetic insecticides.27 Some of the EOs and their bioactive
compounds have already been approved as GRAS (Generally
Regarded As Safe) compounds by the U.S. Food and Drug
Administration. Therefore, in the past few decades EOs and
their bioactive compounds have extensively been studied
against stored product insects for their inherent biological
properties such as toxicity,6,7 repellent,8,9 antifeedant, and
ovicidal activities.10,11 However, relatively little attention has
been paid to their speculated in vivo mode of action against the
insect pests of food grains. A perusal of the literature reveals
that most of the commercially available insecticides exert their
toxicity primarily by impairment in the AChE enzyme activity,
octopaminergic receptor, and antioxidative defense system of
insect pests.12,13
Gaultheria procumbens L. (wintergreen) is an aromatic shrub
(family Ericaceae). Its application as an antimicrobial,
MATERIALS AND METHODS
Chemicals and Equipment. All of the chemicals and reagents
used in the study were procured from Sigma Chemical Co. (St. Louis,
MO, USA), Hi-media, and Sisco Research Lab (Mumbai, India). The
tissue grinder (P/XX/81/1-220 V) was from Yorco Instruments Delhi,
India, the gas chromatographmass spectrometer was from
PerkinElmer (Turbomass Gold, USA), the ELISA plate reader from
Spectromax (SPX340), and the spectrophotometer from Shimadzu
(UV-1800).
Insect Culture. Adults of S. oryzae and R. dominica were obtained
from naturally infested wheat grain from the local market of Mysore,
India. The insect pests were reared separately on clean and uninfested
Received: August 3, 2015
Revised: November 7, 2015
Accepted: November 11, 2015

XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b03797

J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry
wheat grain. Two hundred adult insect pests of each were released in
500 g of wheat grain in a Kilner jar capped with muslin cloth to ensure
ventilation. The jar was kept under laboratory conditions at a
controlled temperature of 27 2 C and relative humidity of 70
5%) at the Department of Food Protection and Infestation Control,
CSIR-CFTRI, Mysore, India. After 72 h, the adults were removed, and
the jar was left for 25 days to obtain the adult insects of the same age
for the experiment. Adult insects, 46 days old, were used for each test
parameter.
Essential Oil and Its Chemical Prole (Gas Chromatogra-
phyMass Spectrometry (GC-MS) Analysis). The EO was
obtained by hydrodistillation of the leaves.6 Leaves were thoroughly
washed three times with distilled water and subjected to hydro-
distillation (3 h) in a Clevengers apparatus. The chemical prole of G.
procumbens EO was analyzed by GC-MS analysis. The GC apparatus
used was a PerkinElmer equipped with a Turbo mass Gold mass
spectrometer. Software was Turbo mass version 5.4.2. The injection
volume of EO was 2 L (1:50 diluted in acetone). The separation was
made on a PerkinElmer Elite-5 column (column length = 30 m, inner
diameter = 0.25 mm, lm thickness = 0.25 mm). The analysis was
carried out under the following conditions: oven, initial temperature =
35 C for 2 min, ramp at 2 C/min to 70 C, hold for 2 min, ramp at 1
C/min to 90 C, hold for 0 min, ramp at 3 C/min to 250 C; inj =
250 C; split =20:1; carrier gas = He; solvent delay = 4.00 min;
transfer temperature = 200 C; source temperature = 180 C; scan,
40400 Da. EO components were identied by comparison of their
retention indices (RI) relative to (C8C22) n-alkanes with those of
authentic compounds and by matching of their mass spectral peaks
available with Wiley, NIST, and NBS mass spectral libraries or with
published data in the literature.19
Insecticidal Activity of EO and MS against S. oryzae and R.
dominica (Fumigation Bioassay). The fumigant toxicity of G.
procumbens EO and its major compound MS was determined via
impregnated paper assays following the method of Shukla et al.11 with
slight modications. Appropriate doses of EO and MS were applied
separately on the lter papers (Whatman no. 1, 3 cm diameter), to
achieve nal concentrations between 1 and 10 L/L air for R. dominica
and between 10 and 200 L/L air for S. oryzae without using any
solvent and attached to the undersurface of lids of desiccator with
volume (2.5 L).11 A control set was kept parallel to the experiments
without any treatments. Thereafter, 50 adults (46 days old) of both
test insect pests were introduced in the desiccator used as exposure
chambers with an appropriate wheat sample as a food source.
Desiccators caps were made airtight using grease to avoid the
accidental release of EO and MS. Mortality of test insect pests was
determined after 24 h of exposure. Knocked down insects were
regarded as dead if they were unable to respond after exposure to heat
from a 60 W lamp. Percent corrected mortality was calculated
following Abbots formula for natural mortality in untreated controls:20
P = T C /100 C 100
P = % corrected mortality, T = % killed in treatment, and C = % killed
in control.
The acute LC50 values of EO and MS were determined using Probit
analysis.21
Antifeedant Activity. The experiment was performed following
the method given by Shukla et al.11 The requisite amounts of EO and
MS were soaked separately in a lter paper and stuck on the inside of
the lid of a glass jar (1 L) to achieve desired concentration (absolute
toxicity = 150 and 5 L/L; LC50 and (1/10 and 1/20 of LC50) for S.
oryzae and R. dominica, respectively). Thereafter, 250 g of uninfested
wheat seed (moisture content = 12.513.5%) was placed inside the
fumigated jars. One hundred adults of each insect pest were released
into the treatment set as well as untreated (control), and the jars were
made airtight using grease and adhesive tapes to avoid water loss from
the seed samples. Wheat seeds with 100 insects of each and without
any treatment were used as a control set. Thereafter, samples were
kept in a temperaturehumidity control cabinet (27 2 C and 80
5% relative humidity) for 6 months. The ecacy of EO and MS was
determined on the basis of percent grain damage and percent weight
Article
loss of treated and untreated (control) seeds after 6 months of storage.
The wheat grain damage was assessed by observing emergence holes
on the surface of the wheat seeds.
The weight loss (%) of the samples was calculated on a fresh weight
basis
weight loss = W1 W /W1 100
where W1 is the weight of the wheat seeds before the experiment and
W is the weight of the wheat seeds after 6 months of the storage
period.
The antifeedant action of the EO and MS as fumigants was
observed by calculating the feeding deterrent index (FDI, %)
FDI = C T /C 100
where C is the weight loss in control samples and T is the weight loss
in treated samples.
Eect of Lethal and Sublethal Exposure of EO and MS on
Biochemical Responses in Test Insect Pests. Preparation of
Enzyme Extract. Insect pests were exposed to lethal and sublethal
doses of EO and MS (LC50 and (1/10 and 1/20 of LC50)) for 24 h of
incubation at room temperature (27 2 C). After the incubation
period, the live insect pests were removed from the desiccator and
homogenized in phosphate buer (pH 7.4, 100 mmoL/L) in a glass
Teon homogenizer. The homogenate was centrifuged at 10000 rpm
at 4 C for 10 min, and the supernatant was stored on ice for
determination of enzyme activity (except cellular glutathione contents
assay).
Acetylcholinesterase (AChE) Activity. AChE activities were
determined in the supernatant using a microplate reader following
the method of Ellmanet al.22 and Galgani and Bocquene.23 The
reaction mixture containing a suitable amount of supernatant and
DTNB in Tris-HCl buer (100 mmoL/L, pH 8.0) was prepared.
Thereafter, acetylthiocholine iodide (ATCI) (10 L of 0.1moL/L
solution) was added to the reaction mixture, and change in absorbance
was monitored over 3 min at 405 nm with a microplate reader. A
change of 0.001 unit of absorbance per minute was considered as 1
unit of the enzyme, and the results were expressed as units per
milligram of protein.
Eect of EO and MS on Oxidative Stress Response Systems.
Insect pests exposed to EO and MS at various doses (LC50 and 1/10
and 1/20 LC50) were used for the measurement of the oxidative stress
markers after 24 h of exposure. The supernatant was used for
biochemical analysis.
Superoxide Dismutase Activity. SOD activity was measured
following the method of Kostyuk and Potapovich.24 The reaction
mixture contained 0.016 moL/L phosphate buer, pH 10; 0.8 mmoL/
L N,N,N,N-tetramethylenediamine, and 0.08 mmoL/L EDTA in a
total volume of 3.0 mL. Thereafter, 0.1 mL of quercetin solution (1.5
mg/10 mL) was added to the reaction mixture to start the reaction. A
suitable amount of the supernatant was added to the reaction mixture.
Thereafter, inhibition of quercetin autoxidation (at pH 10) was
monitored at 406, and the results were expressed as units per milligram
of protein. One unit of enzyme is dened as the amount of the enzyme
that inhibits autoxidation of quercetin by 50%.
Catalase Activity. A reaction mixture was prepared using an aliquot
of supernatant equivalent to 250 g of protein along with 25 L of
H2O2 (3%) added to 3 mL of phosphate buer (50m moL/L, pH 7.4).
The decrease in the absorbance of the reaction mixture was measured
at 240 nm for 5 min.25 The enzyme activity was calculated on the basis
of a molar extinction coecient at 43.6/M/cm, and the results were
expressed as micromoles of H2O2 consumed per minute per milligram
of protein.
Cellular Glutathione Content. The cellular glutathione content was
measured following the method of Hissin and Hilf.26 Insect pests
exposed to EO and MS were homogenized in a glassTeon
homogenizer using phosphate buer, pH 8.0, containing EDTA and
5% trichloroacetic acid (TCA), which was used as a protein
precipitant. Thereafter, the homogenate was centrifuged at 10000
rpm and 4 C for 30 min to obtain supernatant for the assay.
B DOI: 10.1021/acs.jafc.5b03797
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Journal of Agricultural and Food Chemistry Article

Reduced Glutathione. GSH content in the homogenate was
determined following the method of Hissin and Hilf26 with slight
modication. The nal assay mixture (2 mL) contained 100 L of
supernatant, 1.8 mL of phosphate buer containing EDTA (1 mmoL/
L), and 100 L of the o-phthalaldehyde (OPT) solution (1 mg/mL
dissolve in methanol). The assay mixture was vortexed thoroughly and
kept at room temperature for 15 min of incubation. Thereafter,
uorescence was determined at 420 nm with the activation at 350 nm.
Oxidized Glutathione. A mixture (500 L of the supernatant was
added to 200 L of 0.04 moL/L NEM) was prepared and incubated
for 30 min at room temperature. Thereafter, 1.8 mL of 0.1 N NaOH
was added to the mixture (100 L) followed by 100 L of the OPT
solution (1 mg/mL dissolved in the methanol) and kept at room
temperature for 15 min of incubation. Thereafter, uorescence was
determined at 420 nm with the activation at 350 nm.
The uorescence measurements were compared to standard curve
values obtained for known concentrations of GSH and GSSG.
Recovery studies have been carried using xed concentrations of GSH
and GSSG, and recovery was between 90 and 95%.
Protein Estimation. Protein content in tissue homogenate was
measured as described by Lowry et al.,27 using bovine serum albumin
(BSA) as the standard.
Statistical Analysis. The experiments were performed in triplicate,
and the data presented are the mean SE. The results were analyzed
by one-way analysis of variance and Tukeys multiple-range tests to
identify signicant dierences in comparison of means. P values of
0.05 were considered signicant. LC50 (the lethal concentration for
50% mortality) was determined by log-probit analysis, and the data
were analyzed by determining chi-square values and degrees of
freedom. The analysis of data was performed with SPSS program
version 16.0 for Windows (SPSS Inc., IBM Corp.).
exhibited 100% mortality at the same concentrations, 150 and
5.0 L/L, against S. oryzae and R. dominica, respectively,
following 24 h of exposure. Their respective LC50 values are
summarized in Table 2.
Antifeedant Activity. At LC50 and onward concentrations,
EO and MS signicantly protected wheat seeds from insect pest
infestation for up to 6 months of storage. Both EO and MS
caused 100% feeding deterrent index (FDI) at their respective
LC50 doses against S. oryzae and R. dominica. Reductions of
8.26 and 5.33% were recorded in fresh weight of control wheat
seeds infested with S. oryzae and R. dominica, respectively
(Table 3), whereas 1/20 of LC50 doses (2.93 and 3.17 L/L of
EO and MS, respectively, for S. oryzae and 0.14 and 0.10 L/L
of EO and MS, respectively, for R. dominica) had no signicant
eect on antifeedant activity compared to control.
Acetylcholinesterase Activity. A concentration-depend-
ent inhibition of AChE activity was observed in insect pests
exposed to EO and MS. At LC50, maximum inhibitions of
AChE activity of about 20.85 and 13.61% for S. oryzae and
27.50 and 21.98% for R. dominica were observed in insect pests
exposed to EO and MS, respectively, for 24 h (Table 4),
whereas 1/20 of LC50 (2.93 and 3.17 L/L of EO and MS,
respectively, for S. oryzae and 0.14 and 0.10 L/L of EO and
MS, respectively, for R. dominica) doses had no eect on AChE
activity.
Antioxidant Defense System. The results showed EO
and MS caused signicant impairment in the antioxidant

RESULTS
Chemical Composition of EO. GC-MS analysis depicted
defense system at all tested concentrations, whereas 1/20 of
LC50 doses (2.93 and 3.17 L/L of EO and MS, respectively,
for S. oryzae and 0.14 and 0.10 L/L of EO and MS,
the presence of 50 compounds in test EO. MS (retention time, respectively, for R. dominica) had no signicant eect on test
29.934) was identied as the major compound comprising parameters compared to control.
96.61% of the EO (Table 1). The compounds having Superoxide Dismutase. A statistically signicant elevation
percentages of >0.05% of EO are presented in Table 1, in SOD activity was observed compared to controls in S. oryzae
whereas the remaining compounds, ranging between 0.002 and and R. dominica exposed to EO and MS. At LC50 SOD activity
0.05%, are not shown in the table. was elevated by (19.60 and 13.61%) and (17.51% and 13.95 %)
compared to the control in EO and MS exposed insect pests S.
Table 1. Chemical Prole of Gaultheria procumbens EOa oryzae and R. dominica, respectively, following 24 h of exposure
(Figure 1).
no. compound tR RI percentage
Catalase. There is a decreasing trend in the CAT activity. At
1 4-hydroxy-4-methyl-2-pentanone 6.31 854 0.096 LC50 doses signicant decreases in CAT activity (42.48 and
2 -pinene 9.84 935 0.076 32.97% and 39.29 and 28.79%) compared to control were
3 -3-carene 14.27 1004 0.118 observed in EO and MS exposed insect pests S. oryzae and R.
4 methyl salicylate 29.93 1208 96.61
dominica, respectively (Figure 1).
5 Z-citral 33.63 1254 0.735
GSH, GSSG, and GSH/GSSH. The concentrations of GSH
6 citral 36.86 1291 0.916
and GSSG and the GSH/GSSG ratio in insect pests exposed to
7 diethyl phthalate 58.65 1598 0.452
a
EO and MS compared with control are displayed in Figure 2.
tR, retention time; RI, retention index (based on n-alkane series). Treatment sets had higher GSSG and lower GSH and GSH/
GSSG ratios compared to control. At the LC50 value, maximum
Fumigant Toxicity. Both EO and MS showed prominent decreases in GSH/GSSH ratio level (37.34 and 12.84% and
insecticidal activity against the test insect pests. EO and MS 54.09 and 36.51%) compared to control were observed in EO

Table 2. Fumigant Toxicity of G. procumbens EO and MS against S. oryzae and R. dominica after 24 h
insect pest test sample LC50a LC90a slope SE chi square (2) degrees of freedom
S. oryzae EO 58.62 (51.8565.47) 89.79 (78.23114.12) 6.920 0.42 5.68 4
MS 63.49 (56.0971.61) 110.82 (93.89147.54) 5.298 0.33 4.64 3

R. dominica EO 2.71 (2.363.03) 4.23 (3.775.15) 6.469 0.45 2.55 3

MS 1.90 (1.482.29) 3.64 (2.984.94) 4.552 0.31 2.98 3

a
Units LC50 = L/L air; LC90 = L/L air applied for 24 h; 95% lower and upper ducial limits are shown in parentheses.

C DOI: 10.1021/acs.jafc.5b03797
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article

Table 3. Antifeedant Activity of EO and Its Major Compound MS against S. oryzae and R. dominicaa
S. oryzae R. dominica
test sample concn (L/L) % seed damage % wt loss % FDI concn (L/L) % seed damage % wt loss % FDI
control 65.04 2.3 8.26 0.51 76.04 2.7 5.33 0.33

G. procumbens EO 150.0* 0.0 0.0 0.0 0.0 100 5.00* 0.0 0.0 0.0 0.0 100
LC50 0.0 0.0 0.0 0.0 100 LC50 0.0 0.0 0.0 0.0 100
1
/10 59.09 1.8 7.91 0.46 4.23 1
/10 69.04 1.4 5.02 0.33 5.81

methyl salicylate 150.0* 0.0 0.0 0.0 0.0 100 5.00* 0.0 0.0 0.0 0.0 100
LC50 0.0 0.0 0.0 0.0 100 LC50 0.0 0.0 0.0 0.0 100
1
/10 61.45 1.4 7.60 0.22 7.99 1
/10 71.58 2.1 4.93 0.46 7.50
a
Concn, concentration; FDI, feeding deterrent index; *, absolute toxicity; values are the mean (n = 3) SE.

Table 4. Eect of EO and MS on Acetylcholine Esterase investigation test EO was chemically characterized by GC-MS
(AChE) Enzyme Activity in S. oryzae and R. dominicaa analysis. A total of 50 compounds were identied, where MS
was recorded as the major one (96.61%). A similar supportive
% inhibition in AChE activity
observation has been reported by Le-Grand et al.;29 Nikolic et
test sample concn S. oryzae R. dominica al.16 reported MS as a major component of Gaultheria species.
G. procumbens EO LC50 20.85 1.97a 27.50 0.79a EO. Thereafter, a comparative study was performed by EO and
1
/10 11.14 2.08b 16.46 0.78b its major compound MS against all of the test parameters to
determine the role of the major compound in toxicity of EO
methyl salicylate LC50 13.61 1.15a 21.98 0.69a
and possible synergism or antagonism activity of minor
1
/10 6.12 1.41b 7.42 2.25b
compounds.
a
Values are the mean (n = 3) SE. Means followed by the same letter Results of fumigant toxicity revealed that both EO and MS
in the same column are not signicantly dierent according to exhibit a strong toxic eect against the test insect pests. R.
ANOVA and Tukeys multiple-comparison tests (P < 0.05).
dominica was recorded as more susceptible to EO and MS as it
and MS exposed insect pests S. oryzae and R. dominica, required lower doses for absolute mortality than the S. oryzae.
respectively (Figure 2). The results showed great similarity with the earlier hypothesis

of Stefanazzi et al.30 and Chopa and Descamps31 that the

DISCUSSION
The ndings of the present investigation revealed EO and MS
as suitable candidates for insecticide formulation against insect
pests of stored food grain. It has been reported that the
biological activity of EOs is related to their inherent bioactive
compounds, so variation in this may alter their toxicity
potential.28 Therefore, in the present study prior to the
toxicity variation of EOs to insect pests is attributed to levels of
susceptibility, metabolic, biochemical, physiological responses,
and morphological dierence (body size, texture, and thickness
of the cuticle). Furthermore, the study suggested that the
insecticidal activity of test EO is signicantly contributed by its
major compound MS. However, sublethal doses of test EO and
MS did not induce any mortality to test insect pests.

Figure 1. Eect of dierent concentrations of EO and MS on SOD and CAT activity: (A) S. oryzae; (B) R. dominica. Values are the mean (n = 4)
SE. Means followed by the same letter in the bar diagram are not signicantly dierent according to ANOVA and Tukeys multiple-comparison tests.

D DOI: 10.1021/acs.jafc.5b03797
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Journal of Agricultural and Food Chemistry
Figure 2. Eect of dierent concentrations of EO and MS on GSH, GSSG, and the ratio GSH/GSSG: (A) S. oryzae; (B) R. dominica. Values are the
mean (n = 4) SE. Means followed by the same letter in the bar diagram are not signicantly dierent according to ANOVA and Tukeys multiple-
comparison tests.
The results of antifeedant activity revealed that EO and MS
signicantly protect wheat samples from insect pest infestation.
In control set (6576%) damage was observed (emergence of
the hole in seed samples) for both test insect pests, whereas EO
and MS exposed samples exhibited absolute protection at LC50
and onward doses.The speculated reason for this could be the
death of remaining adults and developing embryos through
asphyxiation and reduced rates of fecundity, vitality, fertility,
and natality following long exposure periods of test compounds
as has been emphasized by some earlier researchers.6,32 The
ndings revealed that test EO and MS could be used in the
development of plant-based insecticide formulations for the
protection of food grains and commercially available raw
material such as pasta, pet food, dried fruits, and other
foodstus. Although test EO and MS exhibited strong
insecticidal activity, there are certain limitations such as adverse
eects on food matrix components lipid, starch, proteins, and
organoleptic properties that have to be addressed before its
possible application in insecticide formulations. Hence, further
studies are warranted to address these challenges to satisfy
consumer acceptance of EO-treated products.
In toxicological studies, exposure to lethal and sublethal
doses signicantly aected the enzymatic activities and reected
the biochemical/metabolic disturbances of an organism,
resulting in cell death. Therefore, to elucidate the mechanism
underlying the toxicity eect of test EO and MS at lethal and
sublethal doses, their eects on AChE activity and antioxidant
defense system were studied. The inhibition of the AChE
activity has been used as a sensitive biomarker for the toxicity
evaluation of many synthetic insecticides against a variety of
invertebrate species.23,33,34 AChE hydrolyzes the neurotrans-
mitter acetylcholine at cholinergic synapses and alteration in its
activity leads to accumulation of active acetylcholine in synaptic
clefts, thereby disrupting neurotransmission.35 Results revealed
that at LC50 doses the percent inhibition of AChE activity
E DOI: 10.1021/acs.jafc.5b03797
Article
ranged between 13.61 and 27.50% for both EO and MS against
test insect pests. The percent inhibition of AChE activity was
comparatively lower than some of the earlier reported EOs,
terpenes, and prevalent synthetic insecticides.3638 The
speculated reason for this could be because of their low-dose
requirement for in vivo mortality, as it has been reported that
terpenes required high concentrations to exert their eect on
AChE inhibition during in vivo condition.36,39 Similar
supportive results have been reported in the case of carvone,
-pinene, and geraniol, which showed strong insecticidal
activity but are weak inhibitors of AChE.37,40 Results showed
that acetylcholinesterase was not the main site of action for test
EO and MS. Therefore, further research is warranted to
elucidate other modes of action such as octopaminergic system,
hormone, pheromone system, and cytochrome P450 mono-
oxygenase to the insect pest exposed to EO and MS to extend
our knowledge of the target toxicity mechanisms involved.
In addition, oxidative stress has a profound eect on the
enzymatic (SOD and CAT activity) and nonenzymatic (GSH/
GSSG ratio) antioxidative defense system, thereby disturbing
normal physiological processes.41 SOD catalyzes the dismuta-
tion of the superoxide radicals (O2) to H2O and H2O2, which
is detoxied by CAT. Therefore, SODCAT systems are
considered as the rst line of defense against oxygen toxicity in
organisms. The decreased or increased SOD-CAT activities
depend on the intensity, duration, and type of stress
conditions.42 In the present study, a signicant elevation in
SOD activity compared to control is probably an adaptive
response to neutralize the impact of generated ROS. Contrary
to this, a decrease in CAT activity was observed, which could be
due to increased production of superoxide anion radical as has
been previously reported by earlier workers.43,44 The decrease
in CAT activity could induce the accumulation of toxic H2O2 in
the cell, leading to peroxidation of membrane lipids. In
addition, a signicant decrease in the GSH/GSSG ratio (a
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry
biomarker of oxidative damage) was observed, which suggested
impairment in cellular redox status inside the insect pests
exposed to EO. In view of these results, the nding suggested
that the toxicity of EO and MS to test insect pests might be
related to depletion of CAT activity and GSH/GSSH ratio,
leading to the oxidative imbalance.
G. procumbens EO exhibited promising potential as an
insecticidal agent. The ndings revealed the biological activity
of EO was contributed by its major compound, MS. The
luxuriant growth of G. procumbens in tropical and subtropical
countries provides a source of the raw material for extraction of
EO. Furthermore, EO is commercially available worldwide as a
source of natural fragrances and avoring compounds,
especially in the food industries. The Flavor and Extract
Manufacturers Association (FEMA) of the United States
includes G. procumbens EO in the generally recognized as safe
(GRAS) category (EPA),45 which enhances the possibility of its
formulation as an EO-based insecticide.
In conclusion, this is the rst report on the eect of G.
procumbens EO on in vivo acetylcholinesterase activity and the
antioxidant defense system in S. oryzae and R. dominica. The
ndings revealed that the toxicity of EO might be associated
with oxidative imbalance. In view of the strong fumigant
toxicity and antifeedant activity test, EO could be recom-
mended as a potential plant-based insecticide, which is eective
at a lower dosage, can result in reduced costs, and may alleviate
health and environmental risks often associated with
application of synthetic insecticides. However, before drawing
nal conclusions, further studies are necessary for the
development of test EO-based formulations using an advanced
encapsulation technique to improve ecacy, stability, and also
cost eciency.
AUTHOR INFORMATION
Corresponding Author
*(B.P.) Mail: Department of Botany, Banaras Hindu
University, Varanasi 221005, U.P., India. Phone: +91
9794113055. E-mail: bhanubhu08@gmail.com, bhanubhu08@
redimail.com.
Funding
This research work is nancially supported by the Science and
Engineering Research Board (SERB) New Delhi under Young
Scientist Scheme No. SB/YS/LS-313/2013.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
We thank Prof. Ram Rajasekharan, Director, CSIR-CFTRI,
Mysore, India, for his encouragement and support. We also
thank Prof. N. K. Dubey, Banaras Hindu University, Varanasi,
India, for his valuable suggestion.
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G DOI: 10.1021/acs.jafc.5b03797
J. Agric. Food Chem. XXXX, XXX, XXXXXX