Comparison of antibacterial effects and fumigant toxicity of essential oils extracted from different plants

Industrial Crops & Products 124 (2018) 192–200

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Comparison of antibacterial effects and fumigant toxicity of essential oils extracted from different plants ⁎

Xiao-Fang Tua, Fei Hua, , Kiran Thakura, Xiao-Li Lia, Ying-Shuo Zhanga, Zhao-Jun Weia,b, a b

T

⁎

School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, People’s Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Plant essential oils Chemical composition Antibacterial activity Fumigant toxicity

The chemical composition, antibacterial and insecticidal potential of Anise, Peppermint, Clove, Cinnamon, Pepper, Citronella and Camphor essential oils (EOs) extracted by steam distillation from seven different plant species were investigated. GC–MS analysis revealed that terpenes and phenols were the main components of EOs. The different kinds of EOs exerted appreciable antibacterial action against Gram-positive (B. subtilis and S. aureus) as well as Gram-negative (E. coli and S. typhimurium) bacteria. Among EOs, cinnamon EO exhibited the highest antibacterial effect for all the bacterial strains with the lowest MIC ranging from 0.125 to 0.25 mg/mL, the largest inhibition zone and the strongest inhibition of bacterial growth, followed by clove EO. The moderate inhibitory effects were observed for remaining EOs. On the other hand, cinnamon EO, clove EO, and anise EO exhibited significant fumigant toxicity against Sitophilus oryzae at 7.69 μL/L air concentration after 24 and 48 h exposure. Furthermore, the single component analysis showed the LC50 values of 2.90 for eugenol, 5.45 for cinnamaldehyde, and 5.42 μL/L air for estragole extracted from selected EOs (clove, cinnamon, anise), respectively. The results will pave a way for utilization of plant derived EOs as natural food preservatives to counteract food spoilage and pest’s managements.

1. Introduction Food spoilage and food poisoning caused by microbial infection during the harvesting, processing, transportation and storage of foods present an enormous threat to consumers and the development of food industries. The use of chemically synthesized additives in foods to prevent the food spoilage and pathogenic bacteria has been controversial because of their potential to cause respiratory diseases or other health risks (Fleming-Jones and Smith, 2003). Meanwhile, the continuous application of pharmaceutical antibiotics also caused the emergence of bacterial resistance problems in the past decade. With the increasing concern of safety for the addition of synthetic foods preservatives, the use of natural materials as viable alternative antibacterial agents has become an interesting researchable aspect worldwide. On the other hand, the rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), known as the most destructive pest in stored grains, causes huge loss in the quantity and quality of food and the incidences of foodborne diseases occur every year (Haddi et al., 2015; Herrera et al., 2015). At present, the recurrent and extensive application of chemical-based methods to combat the pests such as

phosphine (PH3) led to increased insect resistance in packed food products (Kljajic and Peric, 2006; Ren et al., 2008). Moreover, some chemical fumigants are being prohibited due to their contribution for environmental pollution and harm to human health. Therefore, it is essential to develop novel and safe strategies for identification and characterization of natural plant based antibacterial agents as well as fumigants for their direct application in food industries. Among the plant derived materials, plant essential oil is an important volatile secondary metabolite in plants, which is well known for its high volatility, low residual generation, and very rare resistance problems (Dippel et al., 2014). Plant EOs contain two main components, terpenes and aromatic compounds (Nouri-Ganbalani et al., 2016), which are commercially used as medicines, flavoring agents in many foods, flavor enhancers and insecticides in spices. In the past decade, the antibacterial, antifungal, antioxidant and insecticidal activities of plant EOs have attracted widespread attention (Celia et al., 2013; Smeriglio et al., 2017; Santos et al., 2018). Many reports have pointed out that the plant essential oils have contact and fumigation toxicity (Stefanazzi et al., 2011), repellent activities (Nenaah, 2014; Nerio et al., 2010), anti-feeding activity (Stefanazzi et al., 2011), and

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Corresponding authors at: School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China. E-mail addresses: [email protected] (X.-F. Tu), [email protected] (F. Hu), [email protected] (K. Thakur), [email protected] (X.-L. Li), [email protected] (Y.-S. Zhang), [email protected] (Z.-J. Wei). https://doi.org/10.1016/j.indcrop.2018.07.065 Received 13 May 2018; Received in revised form 20 July 2018; Accepted 26 July 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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2.4. Antibacterial activity

development and growth inhibitory activity (Waliwitiya et al., 2009) against insects. Meanwhile, many plant EOs have also been investigated for their inhibitory effects on the growth of many bacteria (Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis) and fungi (Candida albicans, Cryptococcus neoformans and Trichophyton mentagrophytes) (Burt, 2004; Salgueiro et al., 2010). Generally, EOs harbor broad spectrum in activity due to the presence of complex chemicals as well as diverse mode of action (Dasgupta, 2016). Considering the excellent antibacterial and insecticidal functions, EOs have the high potential to be developed as novel natural additives to increase the food safety and enhance shelf-life of various foods. Nevertheless, after careful consideration of previous literature analysis, it can be concluded that comprehensive evaluation of antibacterial activity and fumigation toxicity of different plant EOs were rarely investigated. In the lieu of above gaps in knowledge, our research hypothesis can be of great interests for food industries as well as for human health concern. Briefly, we identified the main chemical components of the EOs of the seven different plant species through GC–MS analysis. The antibacterial effects of different plant EOs on food-borne spoilage and pathogenic bacteria were compared, besides, fumigation toxicity of these EOs against S. oryzae. On the basis of these antibacterial and insecticidal activities, the obtained data would eventually promote the utilization of selected EOs as an environment friendly industrial preservative.

2.4.1. Bacterial strains To determine the antibacterial effects of EOs, two kinds of bacteria were used in this study such as Gram-negative strains: Escherichia coli and Salmonella typhimurium, and Gram-positive strains: Bacillus subtilis and Staphylococcus aureus. The test strains were incubated in the Luria Broth medium (Beijing Land Bridge Technology Co., Ltd., China) at 37 °C for 16 h to obtain the active cultures (Ma et al., 2018) 2.4.2. Agar diffusion method for antibacterial activity The antimicrobial activity of the tested EOs were evaluated by agar diffusion method as described previously (Mostafa et al., 2018). Plant EOs were dissolved in ethanol to different concentrations (6.25 mg/mL, 12.50 mg/mL, 25.00 mg/mL, 50.00 mg/mL, 100.00 mg/mL, 200.00 mg/mL, 400.00 mg/mL and 800.00 mg/mL) and filtered by a 0.45 μm microporous filter. 10 mL of Nutrient Agar (Beijing Luqiao Science and Technology Co., Ltd.) was added to each Petri dishes. After solidification, 100 μL of the test bacterial suspension (106 CFU/mL) was spread to the Petri plates. Then after, sterile filter paper discs (6.0-mm diameter, 1.0-mm thickness) containing 10 μL of each plant EO was placed on the surface of seeded Petri plates. The Petri plates were kept in a refrigerator at 4 °C for 2 h to allow the diffusion of plant essential oil followed by incubation at 37 °C for 24 h. The filter papers loaded with 10 μL of ethanol was used as control in each plate during this assay. The inhibition zones were evaluated by measuring the diameter of clear inhibition zone around each paper disc using a vernier caliper which was recorded as an indication of antibacterial activity (Li et al., 2018).

2. Material and methods 2.1. Plant materials Seven different species of plant materials such as Anise (Pimpinella anisum), Peppermint (Mentha haplocalyx), Clove (Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum), Pepper (Zanthoxylum bungeanum), Citronella (Cymbopogon nardus), and Camphor (Cinnamomum camphora) were procured from the local market of Hefei city, China. The obtained raw materials were dried at 40 °C and powered for further extraction.

2.4.3. Minimal inhibitory concentration (MIC) The lowest concentration of EOs without visual growth of bacteria for incubating for 24 h was considered as MIC. MIC was determined according to the previously reported method with slight modification (Zhang et al., 2016). Initially, the stock solution of each EO was dissolved in ethanol to obtain the concentration of 8 mg/mL. Subsequently, two-fold serial dilutions of EOs were prepared in sterile autoclaved tryptone soy broth (TSB) medium to obtain a final concentration ranging from 0.0625 to 8 mg/mL. Finally, 100 μL of bacterial suspension was added individually to each tube. All the tubes were placed in a rotary shaker (ZHWY-200B) at 37 °C and 180 rpm for 24 h to observe the growth of the bacteria.

2.2. Extraction of essential oils The EOs were extracted by hydro distillation in a Clevenger-type apparatus for 3 h from all kind of grounded powder according to the previously reported method (Hamdaoui et al., 2018). The water residues were removed from the oil extracts by using anhydrous sodium sulfate. The obtained EOs were preserved at 4 °C until next use.

2.4.4. Growth curves The anti-bacterial effects of EOs were investigated through growth curve determination. For this, four bacteria were treated with the MIC and 1/2 MIC concentrations, respectively. Then after, the cultured broths (Erlenmeyer flask, 100 mL) supplemented with different EOs were incubated in a rotary shaker (ZHWY-200B) (180 rpm) for 24 h at 37 °C followed by absorbance measurement at 600 nm by using micro plate reader (Infinite 200 PRO, Austria) after every 2 h intervals (Zhang et al., 2017).

2.3. Gas chromatography–mass spectrometry analysis of essential oils The obtained EOs were subjected for chemical composition characterization via gas chromatography–mass spectrometry (GC–MS) (Agilent GC-MS 7890) using a DB-5MS column (30 m × 0.25 mm × 0.25 μm). The experimental conditions were as followed: helium was used as a carrier gas (13 psi), oven temperature was programmed to 50 °C for 2 min and raised to 260 °C at a rate of 5 °C/min and held at this temperature for 10 min and the injector temperature was set at 250 °C. The mass spectra were recorded at 70 eV with the mass range of 42–350 m/z with the ion source temperature of 280 °C (Polatoglu et al., 2016). The major constituents of essential oils were identified by comparing their relative retention times with those of authentic samples and matching their mass spectral peaks available with mass spectral library (NIST 14 Mass Spectral and Wiley Registry™ of Mass Spectral Data) or with published data in the literature (Vasantha-Srinivasan et al., 2016).

2.5. Insecticidal activity against Sitophilus oryzae 2.5.1. Insects culturing The Sitophilus oryzae used in the experiment was collected from Hefei grain storage, Hefei, China and maintained without exposure to any insecticides over many generations. The insects were reared on whole rice and kept in the glass container with the controlled conditions (27 ± 1 °C, 70 ± 5% relative humidity, 12:12 h light:dark cycle). All the bioassays were conducted under the constant environmental conditions.

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2.5.2. Fumigant toxicity of essential oils The fumigant toxicities of essential oils against S. oryzae were determined as described by Huang et al. (Huang et al., 2011) with slight modification. Briefly, 30 random adults of S. oryzae were placed into a 500 mL triangular flask. The filter paper discs (2 × 4 cm) containing different concentrations of EOs were fixed to beneath surface of each flask lid with a needle (Liao et al., 2016). The lid was sealed to avoid the leakage of the test EOs and the triangular flask was incubated in the same culturing conditions mentioned above. The insects incubated without the exposure of EO were used as control in this experiment. Each treatment and control were replicated three times for more accuracy of obtained data. The number of dead insects was recorded at 24 h and 48 h after treatment and the corrected mortality was calculated. During mortality evaluation, the insects were considered dead without any leg or antennal movements when touched with a small brush (Nattudurai et al., 2017). The fumigant toxicity of two major individual constituents of the three selected EOs (clove, cinnamon, and anise) against S. oryzae was evaluated at different doses using the fumigation assay described above. The criteria for selecting three EOs were the fumigant activity and content of individual components in EOs.

Table 1 The main components of the essential oils of Anise (Illicium verum), Peppermint (Mentha haplocalyx), Clove (Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum), Pepper (Zanthoxylum bungeanum), Citronella (Cymbopogon nardus), and Camphor (Cinnamomum camphora) analyzed by GC–MS.

2.6. Statistical analysis To calculate the insect mortality percentage, it was transformed into arsine square-root values for analysis of variance (ANOVA) using the software IBM SPSS Statistics 22.0 (SPSS, USA). The mortality mean values were evaluated and separated using Scheffe’s test at significance level of p value < 0.05. For the data analysis, one-way analysis of variance (ANOVA) was performed using the Origin Lab (Origin Pro 8.0) software. 3. Results and discussion 3.1. Chemical composition of essential oils The chemical compositions of Anise (P. anisum), Peppermint (M. haplocalyx), Clove (S. aromaticum), Cinnamon (C. zeylanicum), Pepper (Z. bungeanum), Citronella (C. nardus), and Camphor (C. camphora) essential oils were analyzed by GC–MS as shown in Table1. The main components of essential oils were found to be terpenes and phenols. From the Table 1, it was clear that the chemical composition of different EOs varied significantly. Among them, the main components of the anise EO were anethole (85.245%), and estragole (6.479%); while, the clove oil constituted eugenol (84.036%) as main component, followed by caryophyllene (11.094%), and α-caryophyllene (3.101%). In cinnamon EO, cinnamaldehyde (86.216%), and methyl salicylate (8.923%) were identified as the main compounds. Whereas, dominant components of peppermint EO were mainly terpenes such as l-caryophyllene (18.546%), β-bourbonene (13.050%), β-ylangene (6.250%) and α-bourbonene (5.639%). The compounds identified in different kinds of EOs were confirmed with results of previous studies (Zhang et al., 2016; Mahdavi et al., 2017; Khani, 2012; Prabuseenivasan et al., 2006); however, substantial variations in their identified compounds as well as in their corresponding contents were noticed. Different growth conditions of the plants, genetic factors, chemical forms, harvesting periods, plant nutritional status and other factors may affect the composition of their EOs which can ultimately contribute to above mentioned chemical diversity. 3.2. Antibacterial activity 3.2.1. Agar diffusion method of antibacterial activity The agar diffusion method was used to preliminarily test the antibacterial activity of seven different EOs with varying concentrations on four bacterial strains including two Gram-positive strains and two 194

Peak no.

RT/min

Compounds

Content(%)

Anise 1 2 3 4 5 6

15.005 17.969 19.652 20.567 22.959 30.000

Linalool Estragole P-methoxybenzaldehyde Anethole 4-Methoxyphenylacetone 2-[2]Pyridyl-cyclohexanol

1.247 6.479 1.598 85.245 1.426 1.694

Peppermint 1 4.529 2 4.641 3 5.080 4 5.134 5 5.559 6 5.634 7 5.827 8 5.906 9 6.127 10 6.318 11 6.505 12 7.756

α-bourbonene β-bourbonene β-ylangene l-Caryophyllene β-Copaene α-limulus Germacrene D a-Acoradiene 2,4-Di-tert-butylphenol Cedrene d-Cadinene Eicosapentaenoic

5.639 13.050 6.250 18.546 5.596 2.697 3.239 4.716 2.805 4.570 4.491 1.923

Clove 1 2 3 4

22.319 24.115 24.996 28.039

Eugenol Caryophyllene α-Caryophyllene Caryophyllene oxide

84.036 11.094 3.101 1.513

Cinnamon 1 2 3 4 5 6 7 8

10.020 17.842 19.771 19.899 20.288 21.558 27.667 42.112

1S-α-Pinene Methyl salicylate 4-Phenyl-2-butanol o-Isopropenyltoluene Cinnamaldehyde Benzene, 2-butenyl7-Methyl-1-naphthol Benzene, 2-ethenyl-1,4-dimethyl-

0.638 8.923 0.956 0.834 86.216 0.837 0.712 0.526

Pepper 1 2 3

3.549 4.122 5.008

30.758 12.796 4.472

4

6.133

a-Isosafrole cis-Isoeugenol 1,7-Dimethyl-7-(4-methyl-3-pentenyl)-tricyclo [2.2.1.0(2,6)]heptane 2,4-Di-tert-butylphenol

Citronella 1 2 3 4 5 6

3.940 4.124 4.263 4.630 5.841 6.004

13.037 3.571 11.543 12.498 1.790 3.570

7 8 9 10

6.198 6.511 7.011 7.596

11 12 13

8.081 8.636 9.126

Citronellyl acetate Eugenol Geranyl acetate b-Elemen Cedrene (3aS,3bR,4S,7R,7aR)-7-methyl-3methylidene-4-(propan-2-yl)octahydro-1Hcyclopenta[1,3]cyclopropa[1,2]benzene α-muurolene d-Cadinene Hedycaryol 1-Hydroxy-1,7-dimethyl-4-isopropyl-2,7cyclodecadiene t-Muurolol γ-Eudesmol β-Eudesmol

Camphor 1 2 3

3.549 4.518 5.008

25.047 4.342 11.936

4 5 6 7 8

5.073 5.141 5.164 5.637 7.096

Safrole α-Copaene 1,7-Dimethyl-7-(4-methyl-3-pentenyl)-tricyclo [2.2.10(2,6)]heptane d-Longifolene l-Caryophyllene (-)-b-Santalene a-Caryophyllene Nerolidol

17.385

3.511 13.658 17.134 2.478 2.283 1.148 3.061

13.394 4.456 4.240 2.389 11.221

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Fig. 1. The inhibition zone (mm) of essential oils. (a) E. coli, (b) S. typhimurium, (c) B. subtilis, (d) S. aureus. All the data are expressed as means ± SD of three independent experiments. Statistical analysis was performed using one−way ANOVA at p < 0.05 designated by superscripts a–f. The ethanol used for dissolving the EOs was taken as a vehicle control for anti−bacterial assay.

essential oils, such as cinnamon and clove, presented a wide range of antibacterial activity in this study, which could be contributed to their chemical composition, the functional groups (alcohols, phenols, aldehydes) presents in major compounds and the synergistic effects between different components (Oussalah et al., 2007). Moreover, after analyzing the main chemical components of these EOs, it was observed that most of the components possessed a benzene ring and a conjugated double bond structure, such as cinnamaldehyde, eugenol and anethole. These structures are known for active electron transfer and interaction with ion channel proteins on the surface of microbial cell membranes which can consequently alter the cell membrane permeability and lead to leakage of cytoplasm and reduced microbial growth (Weerakody et al., 2010; Bajpai et al., 2012; Pavithra et al., 2009). Therefore, from the perspective of composition, benzene rings and conjugated double bond structures as the main components in the plant EOs may be responsible for stronger antibacterial activity which was confirmed in the following experiments.

Gram-negative strains. As shown in Fig. 1, all the tested EOs exhibited appreciable antibacterial activity. As the concentration of EOs increased, the antibacterial activity gradually increased until it reached the maximum level. For the same test organism, cinnamon EO exhibited the strongest antibacterial effect when the concentration of EO reached 800 mg/mL, followed by clove EO. Anise EO showed the weakest antibacterial activity with diameter of inhibition zones less than 14.00 mm. Peppermint, Pepper, Citronella and Camphor EOs were observed with moderate antibacterial activity. Especially for cinnamon EO, when the concentration reached 800 mg/mL, the diameters of inhibition zones for E. coli, S. typhimurium, B. subtilis, S. aureus were 23.50 mm, 24.32 mm, 24.94 mm, and 25.30 mm, respectively. These findings were similar to the previous report (Zhang et al., 2016), which showed that cinnamon EO presented potent inhibitory effects against E. coli and S. aureus with the DIZ (diameter of inhibition zone) values of 19.2 and 28.7 mm. Another study (Prabuseenivasan et al., 2006) also confirmed that cinnamon EO and clove EO proved maximum activity against six bacterial species tested among twenty-one selected essential oils; while, anise EO failed to effectively inhibit Pseudomonas vulgaris, P. aeruginosa and E. coli. In this study, the result revealed non-significant difference in vulnerability between Gram-negative and Gram-positive bacteria for essential oils which was consistent with those reported previously (Prabuseenivasan et al., 2006; El-Maatia et al., 2016). In addition,

3.2.2. Minimal inhibitory concentration It can be clearly concluded from Table 2 that different EOs exhibited different MIC ranges (0.125 mg/mL–4 mg/mL) (Wei et al., 2018). According to the MIC analysis, among all tested samples, strong antibacterial effects of cinnamon EO was observed against E. coli, S. typhimurium, B. subtilis, S. aureus with MIC values ranging from 0.125 to 195

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Table 2 Minimum inhibitory concentration (MIC) of essential oils against tested bacteria. Bacteria strains

S. aureus E. coli B. subtilis S. typhimurium

MIC (mg/mL) of essential oil Camphor

Cinnamon

Peppermint

Citronella

Anise

Pepper

Clove

2 4 4 4

0.125 0.25 0.125 0.125

4 4 4 2

4 2 4 4

2 1 2 0.5

4 1 4 2

1 0.5 1 0.5

Fig. 2. The MIC of all essential oils against test organisms. The middle was growth control (ethanol), the left and the right represent essential oils.

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Fig. 3. The bacterial growth curve with essential oil solutions (1/2 MIC and MIC) at 600 nm. (a) E. coli, (b) S. typhimurium, (c) B. subtilis, (d) S. aureus.

concentration was also different. The time taken to attain the maximum growth was longer in the treatment groups as compared to control which indicated the better inhibition activity of essential oils by delaying the active growth of test bacteria. The findings demonstrated that different incubation time and EO concentration had a great influence on the antibacterial activity which was consistent with previous report (Huang et al., 2018).

0.25 mg/mL, followed by clove EO with MIC ranging from 0.5 to 1 mg/ mL, which was associated with the agar diffusion test. Lv et al. also confirmed that cinnamon EO showed significant antibacterial effects against S. aureus, B. subtilis, E. coli and S. cerevisiae with the MIC value ranging from 0.1 to 0.4 μL/mL (Lv et al., 2011). Another study revealed that cinnamon EO showed maximum activity with MIC values ranging from 0.8 to 3.2 mg/mL followed by clove oil with MIC values ranging from 1.6 to 6.4 mg/mL against all the tested strains (Prabuseenivasan et al., 2006). Meanwhile, Fig. 2 showed the inhibition zones for all the plant EOs against four test organisms at the MIC concentration, which was in accordance with the antibacterial activity order of the agar diffusion assay.

3.3. Insecticidal activity against S. oryzae Fumigation assay was conducted to investigate the toxicity of plant derived EOs against adults of S. oryzae. It was found that among all the EOs, clove and cinnamon EO showed 100% fumigation toxicity at 7.69 μL/L air concentration, followed by anise EO with the mortality of 65.52% and 67.86% after 24 and 48 h fumigant (Fig. 4). Other EOs showed moderate or weak fumigant toxicity at the concentrations of 7.69 μL/L air. These results were in agreement with the previous findings (Lee et al., 2008; Huang et al., 2002), which reported the insecticide effects of clove and cinnamon oils and their major constituents against S. zeamais. At the same dose, a slight progressive fumigation toxicity was observed over a period of 24–48 h. The fumigation toxicity of three selected EOs (cinnamon, clove and anise EO) against S. oryzae was also tested and their LC50 values were presented in Table 3. In the fumigation assay, similar to the previous study (Liao et al., 2016), three selected EOs showed strong toxicity to S. oryzae and resulted into a dose-dependent mortality. In addition, fumigation toxicity was also affected by exposure time. After 24 h of fumigation exposure, the LC50 values of cinnamon, clove and anise oil were 6.693, 3.308 and 4.518 μL/L air, respectively. On the other hand, after EO treatment for 48 h, the LC50 values were 5.195, 2.192, and 3.875 μL/L air, respectively. It reflected that the toxicity of all test essential oils caused higher

3.2.3. Growth curves In order to further confirm the antibacterial activity of different EOs against the four bacteria, the optical density (OD) of the liquid growth medium at 600 nm after treatment with different EOs was measured in this study. The bacterial growth curves generally consist of four stages: lag phase, exponential phase, stationary phase, and decline phase (Yu et al., 2001). As shown in Fig. 3, compared to the EO treatment groups, the number of bacteria in the control group increased rapidly. When the concentration of each EO was at the MIC dose, the OD value of the culture medium was not change after 24 h of culture, indicating that the bacterial growth was almost inhibited. On the other hand, the treatment of different EOs at the 1/2 MIC concentration significantly increased the lag time, suggesting that the normal growth of the bacteria was inhibited to some extent. Among all the EOs, cinnamon EO showed the lowest OD for all the tested strains at the same time, representing the strongest inhibitory activity. In contrast, anise EO showed the weakest inhibitory effect to control the bacterial growth. The time required for different oils to reach the maximum OD at the 1/2 MIC 197

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which were the main compounds of cinnamon EO showed appreciable activity with the LC50 value of 5.456 and 3.227 μL/L air, respectively. Cinnamaldehyde in cinnamon EO and estragole in anise EO displayed similar fumigant toxicity against S. oryzaes adults. These results provided evidence that these three EOs have remarkable fumigant toxicity against adults of S. oryzae when tested individually; however, the final content of the individual components in EOs was not enough to regulate the maximum inhibition as compared to their synergistic effects. As known previously, the insecticidal activities of EOs with aldehyde, ketone, phenol, or alcohol functional groups were more remarkable than those of monoterpenes belonging to hydrocarbons (Kim et al., 2015). The difference observed for fumigant toxicity as compared to previous studies might owe to different strains of S. oryzae, the type and concentration of the EO constituents, diverse species, season, location, climate, soil type, age of the leaves, drying and the extraction method of the EOs. The effective fumigation toxicity of EOs was mainly due to the presence of the volatile components such as terpenes. In this study, the main components of cinnamon, clove and anise EOs were reported as eugenol, cinnamaldehyde and estragole. These compounds exert their neurotoxic effects on insects involving various mechanisms such as GABA, octopamine synapses and inhibition of nerve conduction enzyme acetylcholinesterase (Regnaultroger et al., 2012). Eugenol was reported to exhibit significant insecticidal activity (Dayan et al., 2009) by destroying the octopamine energy (or through its precursor tyramine) and affecting the nervous system of insect (Enan, 2005a,b; Kostyukovsky et al., 2002; Correa et al., 2015). Previous study proved that the exposure to cinnamon and clove EOs resulted into significant reduction of the respiratory rates of maize weevil S. zeamais which was related to the regulation of oxidative phosphorylation processes as well as the regulation of respiration (Gromyszkalkowska and Szubartowska, 1994; Vidau et al., 2009; Nicodemo et al., 2014; Corrêa et al., 2014). In addition, many terpenoid compounds also worked on acetylcholinesterase and the fumigation toxicity against S. zeamais was directly related to acetylcholinesterase activity (Herrera et al., 2015).

Fig. 4. Fumigant toxicity of essential oils extracted from seven plant species against Sitophilus oryzae adults. All the data are expressed as means ± SD of three independent experiments. Statistical analysis was performed using one−way ANOVA at p < 0.05 designated by superscripts a and b.

toxicity after 48 h of exposure than 24 h of exposure. As confirmed from antibacterial results, although anise EO had shown poor activity which was also confirmed by previous studies (Prabuseenivasan et al., 2006); nevertheless, it has shown considerable fumigant toxicity which was compared with previous report which demonstrated its LC50 value of 1.9 μL/L against adults of Culex quinquefasciatus (Pavela, 2014). We also estimated two major constituents individually from three selected EOs against S. oryzae. As shown in Table 4, for clove EO, eugenol was the more potent component with the LC50 value of 2.907 μL/L air which was significantly more active than caryophyllene with the high LC50 value (29.889 μL/L air). Both cinnamaldehyde and methyl salicylate Table 3 Fumigant toxicity of three selected essential oils against Sitophilus oryzaes adults. Essential oil

Exposure time (h)

95% FLb (μL/L air)

LC50a (μL/L air)

Lower

Upper

Slopec ± SE

(χ2)d

Cinnamon

24 48

6.693 5.195

5.228 3.891

9.690 7.058

1.692 ± 0.282 1.680 ± 0.189

4.619 2.563

Clove

24 48

3.308 2.192

2.538 1.582

4.352 2.912

2.039 ± 0.291 1.831 ± 0.132

3.109 4.797

Anise

24 48

4.518 3.875

4.024 3.488

5.110 4.309

4.009 ± 0.311 4.961 ± 0.373

3.182 3.700

a b c d

50% of lethal concentration. Fiducial limits. Slope of the concentration-inhibition regression line ± SE. Chi-square value.

Table 4 Fumigant toxicity of six major compounds from three selected essential oils against Sitophilus oryzaes adults. Essential oils

Compounds

LC50 (μL/L air)

95% FL (μL/L air) Lower

Upper

Slope ± SE

χ2

Clove

Eugenol Caryophyllene

2.907 29.889

2.547 26.251

3.321 33.533

4.215 ± 0.546 4.062 ± 0.617

6.091 2.259

Cinnamon

Cinnamaldehyde Methyl salicylate

5.456 3.227

2.708 1.860

11.931 5.609

4.089 ± 0.568 3.384 ± 0.449

19.714 6.364

Anise

Estragole Anethole

5.421 8.230

3.937 6.568

7.771 10.366

3.177 ± 0.481 3.536 ± 0.338

7.098 12.329

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4. Conclusions

Haddi, K., Mendonca, L.P., Santos, M.F.D., Guedes, R.N.C., Oliveira, E.E., 2015. Metabolic and behavioral mechanisms of indoxacarb resistance in Sitophilus zeamais (Coleoptera: Curculionidae). J. Econ. Entomol. 108, 362. Hamdaoui, A.E., Msanda, F., Boubaker, H., Leach, D., Bombarda, I., Vanloot, P., Aouad, N.E., Abbad, A., Boudyach, E.H., Achemchem, F., Elmoslih, A., Ait Ben Aoumar, A., Mousadik, A.E., 2018. Essential oil composition, antioxidant and antibacterial activities of wild and cultivated Lavandula mairei Humbert. Biochem. Syst. Ecol. 76, 1–7. Herrera, J.M., Zunino, M.P., Dambolena, J.S., Pizzolitto, R.P., Ganan, N.A., Lucini, E.I., Zygadlo, J.A., 2015. Terpene ketones as natural insecticides against Sitophilus zeamais. Ind. Crops Prod. 70, 435–442. Huang, Y., Ho, S.H., Lee, H.C., Yap, Y.L., 2002. Insecticidal properties of eugenol, isoeugenol and methyleugenol and their effects on nutrition of Sitophilus zeamais Motsch. (Coleoptera: Curculionidae) and Tribolium castaneum, (Herbst) (Coleoptera: Tenebrionidae). J. Stored Prod. Res. 38, 403–412. Huang, Y.Z., Hua, H.X., Li, S.G., Yang, C.J., 2011. Contact and fumigant toxicities of calamusenone isolated from Acorus gramineus rhizome against adults of Sitophilus zeamais and Rhizopertha dominica. Insect Sci. 18, 181–188. Huang, J., Qian, C., Xu, H., Huang, Y., 2018. Antibacterial activity of Artemisia asiatica essential oil against some common respiratory infection causing bacterial strains and its mechanism of action in Haemophilus influenz. Microb. Pathog. 114, 470–475. Khani, M., 2012. Insecticidal effects of peppermint and black pepper essential oils against rice weevil, Sitophilus oryzae L. and rice moth, Corcyra cephalonica (St.). J. Med. Plants 11, 97–110. Kim, S.W., Lee, H.R., Jang, M.J., Jung, C.S., Park, I.K., 2015. Fumigant toxicity of Lamiaceae plant essential oils and blends of their constituents against adult rice weevil Sitophilus oryzae. Molecules 21, 361. Kljajic, P., Peric, I., 2006. Susceptibility to contact insecticides of granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae) originating from different locations in the former Yugoslavia. J. Stored Prod. Res. 42, 149–161. Kostyukovsky, M., Rafaeli, A., Gileadi, C., Demchenko, N., Shaaya, E., 2002. Activation of octopaminergic receptors by essential oil constituents isolated from aromatic plants: possible mode of action against insect pests. Pest Manage. Sci. 58, 1101–1106. Lee, E.J., Kim, J.R., Choi, D.R., Ahn, Y.J., 2008. Toxicity of cassia and cinnamon oil compounds and cinnamaldehyde-related compounds to Sitophilus oryzae (Coleoptera: Curculionidae). J. Econ. Entomol. 101, 1960–1966. Li, L., Thakur, K., Liao, B.Y., Zhang, J.G., Wei, Z.J., 2018. Antioxidant and antimicrobial potential of polysaccharides sequentially extracted from Polygonatum cyrtonema Hua. Int. J. Biol. Macromol. 114, 317–323. Liao, M., Xiao, J.J., Zhou, L.J., Liu, Y., Wu, X.W., Hua, R.M., Wang, G.R., Cao, H.Q., 2016. Insecticidal activity of Melaleuca alternifolia essential oil and RNA-Seq analysis of Sitophilus zeamais transcriptome in response to oil fumigation. PLoS One 11, e0167748. Lv, F., Liang, H., Yuan, Q., Li, C., 2011. In vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms. Food Rev. Int. 44, 3057–3064. Ma, Y.L., Zhu, D.Y., Thakur, K., Wang, C.H., Wang, H., Ren, Y.F., Zhang, J.G., Wei, Z.J., 2018. Antioxidant and antibacterial evaluation of polysaccharides sequentially extracted from onion (Allium cepa L.). Int. J. Biol. Macromol. 111, 92–101. Mahdavi, V., Hosseini, S.E., Sharifan, A., 2017. Effect of edible chitosan film enriched with anise (Pimpinella anisum L.) essential oil on shelf life and quality of the chicken burger. Food Sci. Nutr. 6, 269–279. Mostafa, A.A., Al-Askar, A.A., Almaary, K.S., Dawoud, T.M., Sholkamy, E.N., Bakri, M.M., 2018. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi J. Biol. Sci. 25, 361–366. Nattudurai, G., Baskar, K., Paulra, M.G., Islam, V.I., Ignacimuthu, S., Duraipandiyan, V., 2017. Toxic effect of Atalantia monophylla essential oil on Callosobruchus maculatus and Sitophilus oryzae. Environ. Sci. Pollut. Res. 24, 1–11. Nenaah, G.E., 2014. Chemical composition, insecticidal and repellence activities of essential oils of three Achillea species against the Khapra beetle (Coleoptera: Dermestidae). J. Pest Sci. 87, 273–283. Nerio, L.S., Oliveroverbel, J., Stashenko, E., 2010. Repellent activity of essential oils: a review. Bioresour. Technol. 101, 372–378. Nicodemo, D., Maioli, M.A., Medeiros, H.C., Guelfi, M., Balieira, K.V., De, J.D., Mingatto, F.E., 2014. Fipronil and imidacloprid reduce honeybee mitochondrial activity. Environ. Toxicol. Chem. 33, 2070–2075. Nouri-Ganbalani, G., Ebadollahi, A., Nouri, A., 2016. Chemical composition of the essential oil of Eucalyptus procera Dehnh. and its insecticidal effects against two stored product insects. J. Essent. Oil Bear. Plants 19, 1234–1242. Oussalah, M., Caillet, S., Saucier, L., Lacroix, M., 2007. Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control 18, 414–420. Pavela, R., 2014. Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna. J. Asia-Pac. Entomol. 17, 287–293. Pavithra, P.S., Sreevidya, N., Verma, R.S., 2009. Antibacterial activity and chemical composition of essential oil of Pamburus missionis. J. Ethnopharmacol. 124, 151–153. Polatoglu, K., Karakoc, C., Yücel, Y.Y., Gücel, S., Demirci, B., Baser, K.H.C., Demirci, F., 2016. Insecticidal activity of edible Crithmum maritimum L. essential oil against Coleopteran and Lepidopteran insects. Ind. Crops Prod. 89, 383–389. Prabuseenivasan, S., Jayakumar, M., Ignacimuthu, S., 2006. In vitro antibacterial activity of some plant essential oils. BMC Complement. Altern. Med. 6, 39. Regnaultroger, C., Vincent, C., Arnason, J.T., 2012. Essential oils in insect control: lowrisk products in a high-stakes world. Annu. Rev. Entomol. 57, 405. Ren, Y.L., Mahona, D., Gravera, J.V.S., Head, M., 2008. Fumigation trial on direct

In this study, terpenes and phenols were identified as the main chemical constituents of EOs derived from seven different plant species. The EOs especially cinnamon EO and clove EO had demonstrated important antimicrobial activity against four tested food borne bacteria. The antibacterial activity increased with the increasing EO concentration and exposure time. Meanwhile, among all the tested EOs, cinnamon, clove and anise EO also displayed significant fumigant toxicity against the most common food grains pest S. oryzae. The LC50 values of 2.90 for eugenol, 5.45 for cinnamaldehyde, and 5.42 μL/L air for estragole extracted from selected EOs (clove, cinnamon, anise), respectively reveal their remarkable fumigant potential. On the basis of potent antibacterial and fumigant toxicity, the two EOs namely cinnamon EO and clove EO will be further studied for their underlying mechanisms and synergetic effects. To conclude, the selected EOs can be used as industrial preservatives to control the spoilage and deterioration of stored grains. Conflict of interest There is none to declare. Acknowledgments This study was supported by the Major Projects of Science and Technology in Anhui Province (17030701058, 17030701028, and 17030701024), and the National Natural Science Foundation of China (31301657). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.07.065. References Bajpai, V.K., Baek, K.H., Kang, S.C., 2012. Control of Salmonella in foods by using essential oils: a review. Food Rev. Int. 45, 722–734. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94, 223–253. Celia, C., Trapasso, E., Locatelli, M., Navarra, M., Ventura, C.A., Wolfram, J., Carafa, M., Morittu, V.M., Britti, D., Marzio, L.D., Paolino, D., 2013. Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells. Colloid Surf. B 112, 548–553. Corrêa, A.S., Tomé, H.V.V., Braga, L.S., Martins, G.F., de Oliveira, L.O., Guedes, R.N.C., 2014. Are mitochondrial lineages, mitochondrial lysis and respiration rate associated with phosphine susceptibility in the maize weevil Sitophilus zeamais? Ann. Appl. Biol. 165, 137–146. Correa, Y.D.C.G., Faroni, L.R.A., Haddi, K., Oliveira, E.E., Pereira, E.J.G., 2015. Locomotory and physiological responses induced by clove and cinnamon essential oils in the maize weevil Sitophilus zeamais. Pestic. Biochem. Phys. 125, 31. Dasgupta, M., 2016. Chemical composition, insecticidal and biochemical effects of essential oils of different plant species from Northern Egypt on the rice weevil, Sitophilus oryzae L. J. Pest Sci. 89, 219–229. Dayan, F.E., Cantrell, C.L., Duke, S.O., 2009. Natural products in crop protection. Bioorg. Med. Chem. 17, 4022–4034. Dippel, S., Oberhofer, G., Kahnt, J., Gerischer, L., Opitz, L., Schachtner, J., Stanke, M., Schütz, S., Wimmer, E.A., Angeli, S., 2014. Tissue-specific transcriptomics, chromosomal localization, and phylogeny of chemosensory and odorant binding proteins from the red flour beetle Tribolium castaneum reveal subgroup specificities for olfaction or more general functions. BMC Genom. 15, 1–14. El-Maatia, M.F.A., Mahgoubb, S.A., Labiba, S.M., Al-Gabya, A.M.A., Ramadana, M.F., 2016. Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities. Eur. J. Integr. Med. 8, 494–504. Enan, E.E., 2005a. Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to plant essential oils. Arch. Insect Biochem. Physiol. 59, 161–171. Enan, E.E., 2005b. Molecular response of Drosophila melanogaster tyramine receptor cascade to plant essential oils. Insect Biochem. Mol. Biol. 35, 309–321. Fleming-Jones, M.E., Smith, R.E., 2003. Volatile organic compounds in foods: a five year study. J. Agric. Food Chem. 51, 8120–8127. Gromyszkalkowska, K., Szubartowska, E., 1994. Respiratory metabolism of millipedes after poisoning with cypermethrin. Bull. Environ. Contam. Toxicol. 53, 765–770.

199

Industrial Crops & Products 124 (2018) 192–200

X.-F. Tu et al.

Vidau, C., Brunet, J.L., Badiou, A., Belzunces, L.P., 2009. Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2. Toxicol. In Vitro 23, 589–597. Waliwitiya, R., Kennedy, C., Lowenberger, C., 2009. Larvicidal and oviposition altering activity of monoterpenoids, trans-anethole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae). Pest Manag. Sci. 65, 241–248. Weerakody, N.S., Caffin, N., Turner, M.S., Dykes, G.A., 2010. In vitro antimicrobial activity of less-utilized spice and herb extracts against selected food-borne bacteria. Food Control 21, 1408–1414. Wei, Z.F., Zhao, R.N., Dong, L.J., Zhao, X.Y., Su, J.X., Zhao, M., Li, L., Bian, Y.J., Zhang, L.J., 2018. Dual-cooled solvent-free microwave extraction of Salvia officinalis L. essential oil and evaluation of its antimicrobial activity. Ind. Crops Prod. 120, 171–176. Yu, J.Y., McGenity, T.J., Coleman, M.L., 2001. Solution chemistry during the lag phase and exponential phase of pyrite oxidation by Thiobacillus ferrooxidans. Chem. Geol. 175, 307–317. Zhang, Y.B., Liu, X.Y., Wang, Y.F., Jiang, P.P., Quek, S.Y., 2016. Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control 59, 282–289. Zhang, L.L., Zhang, L.F., Hu, Q.P., Hao, D.L., Xu, J.G., 2017. Chemical composition, antibacterial activity of Cyperus rotundus rhizomes essential oil against Staphylococcus aureus via membrane disruption and apoptosis pathway. Food Control 80, 290–296.

application of liquid carbonyl sulphide to wheat in a 2500 t concrete silo. J. Stored Prod. Res. 44, 115–125. Salgueiro, L., Martins, A.P., Correia, H., 2010. Raw materials: the importance of quality and safety. Flavour Fragr. J. 25, 253–271. Santos, J.F.S., Rocha, J.E., Bezerra, C.F., Silva, M.K.N., Matos, Y.M.L.S., Freitas, T.S., Santos, A.T.L., Cruz, R.P., Machado, A.J.T., Rodrigues, T.H.S., Brito, E.S., Sales, D.L., Almeida, W.O., Costa, J.G.M., Coutinho, H.D.M., Morais-Braga, M.F.B., 2018. Chemical composition, antifungal activity and potential anti-virulence evaluation of the Eugenia uniflora essential oil against Candida spp. Food Chem. 261, 233–239. Smeriglio, A., Alloisio, S., Raimondo, F.M., Denaro, M., Xiao, J.B., Cornara, L., Trombetta, D., 2017. Essential oil of Citrus lumia Risso: phytochemical profile, antioxidantproperties and activity on the central nervous system. Food Chem. Toxicol. https://doi.org/10.1016/j.fct.2017.12.053. Stefanazzi, N., Teodoro, S., Ferrero, A., 2011. Composition and toxic, repellent and feeding deterrent activity of essential oils against the stored-grain pests Tribolium castaneum (Coleoptera: Tenebrionidae) and Sitophilus oryzae (Coleoptera: Curculionidae). Pest Manag. Sci. 67, 639–646. Vasantha-Srinivasan, P., Senthil-Nathan, S., Thanigaive, A., Edwin, E.S., Ponsankar, A., Selin-Rani, S., Pradeepa, V., Sakthi-Bhagavathy, M., Kalaivani, K., Hunter, W.B., Duraipandiyan, V., Al-Dhabi, N.A., 2016. Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm, Eudrilus eugeniae Kinb. Chemosphere 155, 336–347.

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