Assessment of Toxicity, Antifeedant Activity, and Biochemical

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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, CSIRCentral 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 chromatography−mass 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 μL/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 significant dose-dependent response in all test parameters was observed. The results demonstrated that G. procumbens EO could play a significant 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

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INTRODUCTION Sitophilus oryzae L. (Coleoptera: Curculionidae) and Rhyzopertha dominica F. (Coleoptera: Bostrychidae) are the two foremost insect pest species of food grainswheat, barley, rye, oats, and their finished products. Each of their stages, such as eggs, larvae, and pupae, feed and develop concealed within the seed kernels, causing huge losses to the affected 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 effective biorational and eco-friendly insecticides.2,3 In this prospect, essential oils (EOs) often have strong fumigant action, low mammalian toxicity, reduced effect on nontarget organism, and less/no persistence activity, hence, could be considered as potential biorational alternatives to synthetic insecticides.2−7 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, © XXXX American Chemical Society

antileishmanial, antioxidant, and antidiabetic agent has been well explored in the literature.14−16 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 efficacy 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 profile of G. procumbens EO and its efficacy against the insect pest S. oryzae and R. dominica. In addition, effects 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 effect on AChE enzyme activity and antioxidative defense system (SOD, CAT, and GSH/GSSH).

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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 chromatograph−mass 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

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DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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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, 4−6 days old, were used for each test parameter. Essential Oil and Its Chemical Profile (Gas Chromatography−Mass 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 hydrodistillation (3 h) in a Clevenger’s apparatus. The chemical profile 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 μL (1:50 diluted in acetone). The separation was made on a PerkinElmer Elite-5 column (column length = 30 m, inner diameter = 0.25 mm, film 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, 40−400 Da. EO components were identified by comparison of their retention indices (RI) relative to (C8−C22) 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 modifications. Appropriate doses of EO and MS were applied separately on the filter papers (Whatman no. 1, 3 cm diameter), to achieve final concentrations between 1 and 10 μL/L air for R. dominica and between 10 and 200 μL/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 (4−6 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 Abbot’s formula for natural mortality in untreated controls:20

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. Effect 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 buffer (pH 7.4, 100 mmoL/L) in a glass− Teflon 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 buffer (100 mmoL/L, pH 8.0) was prepared. Thereafter, acetylthiocholine iodide (ATCI) (10 μL 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. Effect 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 buffer, 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 defined 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 μg of protein along with 25 μL of H2O2 (3%) added to 3 mL of phosphate buffer (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 coefficient 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 glass−Teflon homogenizer using phosphate buffer, 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.

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 filter paper and stuck on the inside of the lid of a glass jar (1 L) to achieve desired concentration (absolute toxicity = 150 and 5 μL/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 = 12.5−13.5%) 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 temperature−humidity control cabinet (27 ± 2 °C and 80 ± 5% relative humidity) for 6 months. The efficacy of EO and MS was determined on the basis of percent grain damage and percent weight B

DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Reduced Glutathione. GSH content in the homogenate was determined following the method of Hissin and Hilf26 with slight modification. The final assay mixture (2 mL) contained 100 μL of supernatant, 1.8 mL of phosphate buffer containing EDTA (1 mmoL/ L), and 100 μL 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, fluorescence was determined at 420 nm with the activation at 350 nm. Oxidized Glutathione. A mixture (500 μL of the supernatant was added to 200 μL 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 μL) followed by 100 μL of the OPT solution (1 mg/mL dissolved in the methanol) and kept at room temperature for 15 min of incubation. Thereafter, fluorescence was determined at 420 nm with the activation at 350 nm. The fluorescence measurements were compared to standard curve values obtained for known concentrations of GSH and GSSG. Recovery studies have been carried using fixed 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 Tukey’s multiple-range tests to identify significant differences in comparison of means. P values of ≤0.05 were considered significant. 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 μL/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 significantly 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 μL/L of EO and MS, respectively, for S. oryzae and 0.14 and 0.10 μL/L of EO and MS, respectively, for R. dominica) had no significant effect on antifeedant activity compared to control. Acetylcholinesterase Activity. A concentration-dependent 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 μL/L of EO and MS, respectively, for S. oryzae and 0.14 and 0.10 μL/L of EO and MS, respectively, for R. dominica) doses had no effect on AChE activity. Antioxidant Defense System. The results showed EO and MS caused significant impairment in the antioxidant defense system at all tested concentrations, whereas 1/20 of LC50 doses (2.93 and 3.17 μL/L of EO and MS, respectively, for S. oryzae and 0.14 and 0.10 μL/L of EO and MS, respectively, for R. dominica) had no significant effect on test parameters compared to control. Superoxide Dismutase. A statistically significant elevation in SOD activity was observed compared to controls in S. oryzae and R. dominica exposed to EO and MS. At LC50 SOD activity 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. oryzae and R. dominica, respectively, following 24 h of exposure (Figure 1). Catalase. There is a decreasing trend in the CAT activity. At LC50 doses significant decreases in CAT activity (42.48 and 32.97% and 39.29 and 28.79%) compared to control were observed in EO and MS exposed insect pests S. oryzae and R. dominica, respectively (Figure 1). GSH, GSSG, and GSH/GSSH. The concentrations of GSH and GSSG and the GSH/GSSG ratio in insect pests exposed to EO and MS compared with control are displayed in Figure 2. Treatment sets had higher GSSG and lower GSH and GSH/ GSSG ratios compared to control. At the LC50 value, maximum decreases in GSH/GSSH ratio level (37.34 and 12.84% and 54.09 and 36.51%) compared to control were observed in EO

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RESULTS Chemical Composition of EO. GC-MS analysis depicted the presence of 50 compounds in test EO. MS (retention time, 29.934) was identified as the major compound comprising 96.61% of the EO (Table 1). The compounds having percentages of >0.05% of EO are presented in Table 1, whereas the remaining compounds, ranging between 0.002 and 0.05%, are not shown in the table. Table 1. Chemical Profile of Gaultheria procumbens EOa no.

compound

tR

RI

percentage

1 2 3 4 5 6 7

4-hydroxy-4-methyl-2-pentanone α-pinene δ-3-carene methyl salicylate Z-citral citral diethyl phthalate

6.31 9.84 14.27 29.93 33.63 36.86 58.65

854 935 1004 1208 1254 1291 1598

0.096 0.076 0.118 96.61 0.735 0.916 0.452

a

tR, retention time; RI, retention index (based on n-alkane series).

Fumigant Toxicity. Both EO and MS showed prominent insecticidal activity against the test insect pests. EO and MS

Table 2. Fumigant Toxicity of G. procumbens EO and MS against S. oryzae and R. dominica after 24 h test sample

LC50a

LC90a

slope ± SE

chi square (χ2)

degrees of freedom

S. oryzae

EO MS

58.62 (51.85−65.47) 63.49 (56.09−71.61)

89.79 (78.23−114.12) 110.82 (93.89−147.54)

6.920 ± 0.42 5.298 ± 0.33

5.68 4.64

4 3

R. dominica

EO MS

2.71 (2.36−3.03) 1.90 (1.48−2.29)

4.23 (3.77−5.15) 3.64 (2.98−4.94)

6.469 ± 0.45 4.552 ± 0.31

2.55 2.98

3 3

insect pest

a

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

DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 3. Antifeedant Activity of EO and Its Major Compound MS against S. oryzae and R. dominicaa S. oryzae test sample

concn (μL/L)

control

a

% seed damage

% wt loss

65.04 ± 2.3

8.26 ± 0.51

R. dominica % FDI

concn (μL/L)

% seed damage

% wt loss

76.04 ± 2.7

5.33 ± 0.33

% FDI

G. procumbens EO

150.0* LC50 1 /10

0.0 ± 0.0 0.0 ± 0.0 59.09 ± 1.8

0.0 ± 0.0 0.0 ± 0.0 7.91 ± 0.46

100 100 4.23

5.00* LC50 1 /10

0.0 ± 0.0 0.0 ± 0.0 69.04 ± 1.4

0.0 ± 0.0 0.0 ± 0.0 5.02 ± 0.33

100 100 5.81

methyl salicylate

150.0* LC50 1 /10

0.0 ± 0.0 0.0 ± 0.0 61.45 ± 1.4

0.0 ± 0.0 0.0 ± 0.0 7.60 ± 0.22

100 100 7.99

5.00* LC50 1 /10

0.0 ± 0.0 0.0 ± 0.0 71.58 ± 2.1

0.0 ± 0.0 0.0 ± 0.0 4.93 ± 0.46

100 100 7.50

Concn, concentration; FDI, feeding deterrent index; *, absolute toxicity; values are the mean (n = 3) ± SE.

investigation test EO was chemically characterized by GC-MS analysis. A total of 50 compounds were identified, where MS was recorded as the major one (96.61%). A similar supportive observation has been reported by Le-Grand et al.;29 Nikolic et al.16 reported MS as a major component of Gaultheria species. EO. Thereafter, a comparative study was performed by EO and its major compound MS against all of the test parameters to determine the role of the major compound in toxicity of EO and possible synergism or antagonism activity of minor compounds. Results of fumigant toxicity revealed that both EO and MS exhibit a strong toxic effect against the test insect pests. R. dominica was recorded as more susceptible to EO and MS as it required lower doses for absolute mortality than the S. oryzae. The results showed great similarity with the earlier hypothesis of Stefanazzi et al.30 and Chopa and Descamps31 that the toxicity variation of EOs to insect pests is attributed to levels of susceptibility, metabolic, biochemical, physiological responses, and morphological difference (body size, texture, and thickness of the cuticle). Furthermore, the study suggested that the insecticidal activity of test EO is significantly contributed by its major compound MS. However, sublethal doses of test EO and MS did not induce any mortality to test insect pests.

Table 4. Effect of EO and MS on Acetylcholine Esterase (AChE) Enzyme Activity in S. oryzae and R. dominicaa % inhibition in AChE activity test sample

concn

S. oryzae

R. dominica

G. procumbens EO

LC50 1 /10

20.85 ± 1.97a 11.14 ± 2.08b

27.50 ± 0.79a 16.46 ± 0.78b

LC50 /10

13.61 ± 1.15a 6.12 ± 1.41b

21.98 ± 0.69a 7.42 ± 2.25b

methyl salicylate

1

Values are the mean (n = 3) ± SE. Means followed by the same letter in the same column are not significantly different according to ANOVA and Tukey’s multiple-comparison tests (P < 0.05).

a

and MS exposed insect pests S. oryzae and R. dominica, respectively (Figure 2).

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DISCUSSION The findings 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

Figure 1. Effect of different 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 significantly different according to ANOVA and Tukey’s multiple-comparison tests. D

DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of different 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 significantly different according to ANOVA and Tukey’s multiplecomparison tests.

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.36−38 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 effect on AChE inhibition during in vivo condition.36,39 Similar supportive results have been reported in the case of carvone, β-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 monooxygenase 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 effect on the enzymatic (SOD and CAT activity) and nonenzymatic (GSH/ GSSG ratio) antioxidative defense system, thereby disturbing normal physiological processes.41 SOD catalyzes the dismutation of the superoxide radicals (O2−) to H2O and H2O2, which is detoxified by CAT. Therefore, SOD−CAT systems are considered as the first 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 significant 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 significant decrease in the GSH/GSSG ratio (a

The results of antifeedant activity revealed that EO and MS significantly protect wheat samples from insect pest infestation. In control set (65−76%) 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 findings 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 foodstuffs. Although test EO and MS exhibited strong insecticidal activity, there are certain limitations such as adverse effects 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 significantly affected the enzymatic activities and reflected the biochemical/metabolic disturbances of an organism, resulting in cell death. Therefore, to elucidate the mechanism underlying the toxicity effect of test EO and MS at lethal and sublethal doses, their effects 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 neurotransmitter 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 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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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 finding 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 findings 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 flavoring 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 first report on the effect of G. procumbens EO on in vivo acetylcholinesterase activity and the antioxidant defense system in S. oryzae and R. dominica. The findings 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 recommended as a potential plant-based insecticide, which is effective 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 final conclusions, further studies are necessary for the development of test EO-based formulations using an advanced encapsulation technique to improve efficacy, stability, and also cost efficiency.

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AUTHOR INFORMATION

Corresponding Author

*(B.P.) Mail: Department of Botany, Banaras Hindu University, Varanasi 221005, U.P., India. Phone: +91 9794113055. E-mail: [email protected], bhanubhu08@ rediffmail.com. Funding

This research work is financially 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 financial interest.

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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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DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b03797 J. Agric. Food Chem. XXXX, XXX, XXX−XXX