Insecticidal activity of edible Crithmum maritimum L. essential oil against Coleopteran and Lepidopteran insects

Industrial Crops and Products 89 (2016) 383–389

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Insecticidal activity of edible Crithmum maritimum L. essential oil against Coleopteran and Lepidopteran insects Kaan Polato˘glu a,∗ , Ömer Cem Karakoc¸ b , Yasemin Yücel Yücel c , Salih Gücel d , Betül Demirci e , Kemal Hüsnü Can Bas¸er e,f,g , Fatih Demirci e a

Istanbul Kemerburgaz University, Faculty of Pharmacy, Department of Analytical Chemistry, 34217 Istanbul, Turkey Cankiri Karatekin University, Yaprakli Vocational School, Department of Crop and Animal Protection, 18100 Cankiri, Turkey c Istanbul Kemerburgaz University, Faculty of Pharmacy, Department of Biochemistry, 34217 Istanbul, Turkey d Near East University, Institute of Environmental Sciences, 10, Mersin, Nicosia, Turkey e Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskisehir, Turkey f King Saud University, Faculty of Science & Letters, Department of Botany and Microbiology, 11451 Riyadh, Saudi Arabia g Near East University, Faculty of Pharmacy, Department of Pharmacognosy, 10, Mersin, Nicosia, Turkey b

a r t i c l e

i n f o

Article history: Received 18 January 2016 Received in revised form 18 April 2016 Accepted 18 May 2016 Keywords: Essential oil Storage pests Contact and fumigant toxicity AChE and BChE inhibition Antimicrobial activity

a b s t r a c t Crithmum maritimum L. (Apiaceae) is an edible plant that is used in salads or consumed as pickles in Cyprus. In our insecticidal screening study of the plant species of Cyprus, we have studied the insecticidal activity (contact and fumigant toxicity) of the essential oil of Crithmum maritimum against stored product pests Sitophilus granarius L., S. oryzae L., Tribolium castaneum Herbst., T. confusum Jacquelin du Val., Rhyzopertha dominica Fabricius., Oryzaephilus surinamensis L. (Coleoptera) and the field pest Spodoptera exigua (Hübner) (Lepidoptera). Dried leaves of C. maritimum afforded an essential oil with 0.22% (v/w) yield. The essential oil was analyzed with GC, GC–MS and the major components of the oil were identified as ␥-terpinene (39.3%), ␤-phellandrene (22.6%), carvacrol methylether (10.5%) and (Z)-␤-ocimene (8.2%). Highest contact toxicity of the oil was observed against S. oryzae, R. dominica and O. surinamensis (1 ␮L/insect application of 10% (v/v) oil solution in acetone, after 72 h, 93.30%, 83.26% and 70.33%, respectively). Highest fumigant toxicity was observed for S. granarius, S. oryzae and O. surinamensis (10 ␮L/10 mL container application of 10% (v/v) oil solution in acetone, after 48 h, 100.00%, 100.00% and 90.75%, respectively). Essential oil was tested on S. exigua larvae at different development stages (3rd , 4th and 5th instar larvae). 100 ␮L/mL essential oil was used with 1, 2 and 4 ␮L/larvae concentrations for 3rd , 4th and 5th instar S. exigua larvae, respectively. The toxicity of the oil against the larvae was evaluated after 24 h. C. maritimum essential oil afforded 89% mortality against the 3rd instar larvae however the mortality was decreased to 50% in the 4th and none in the 5th instar S. exigua larvae. The oil also afforded considerable AChE and BChE enzyme inhibition (50.3% and 59.8%, respectively) using the Ellman spectrophotometric method. The essential oil did not produced considerable activity (104 fold lower than the positive controls) against the selected pathogens that could be found on wheat (Bacillus cereus, B. subtilis, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli) using a microdilution method for antimicrobial evaluation. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Environmental concerns and increasing demand for food safety initiated the reconsideration of the current protection strategies of

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (K. Polato˘glu), [email protected] (Ö.C. Karakoc¸), [email protected] (Y. Yücel Yücel), [email protected] (S. Gücel), [email protected] (B. Demirci), [email protected] (K.H.C. Bas¸er). http://dx.doi.org/10.1016/j.indcrop.2016.05.032 0926-6690/© 2016 Elsevier B.V. All rights reserved.

the agricultural production. The use of synthetic pesticides have many environmental concerns including contamination of soil, water (Holoubek et al., 2009; Aharonson et al., 1987), reduction of biodiversity (Flavia et al., 2010), decline of pollinators (Lora et al., 2005; Rafael et al., 2009), effects on non-target species (Sparling et al., 2001; Sparling, Fellers and McConnell, 2001; Fry, 1995; Flexner et al., 1986; Flexner, Lighthart and Croft, 1986) and depletion of ozone layer (Butler and Rodriguez, 1996). Additionally most of the synthetic pesticides have serious toxicity and could cause many health problems to their users as well as the consumers of the agricultural product via the residues on the product or through

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environmental contamination (Zahm and Blair, 1992; Maele-Fabry et al., 2006; Sanborn et al., 2007). Because of the mentioned concerns, many countries have decided to adopt new regulations on the pest management strategies in agricultural production. An example of this is the “European Pesticide Regulation (EC) No. 1107/2009” which supports the use of pesticides that are less harmful or harmless to the health and the environment. Such regulations caused an urge to find new and safer alternatives to the existing synthetic fumigants (Villaverde et al., 2014). Currently, very toxic substances such as phosphine, sulfuryl fluoride, carbonyl sulfide and ethane dinitrile are used in the management of stored product pests. However there is a growing interest on the pest management agents from the natural products. Due to their volatile nature, the essential oils could be regarded as a possible alternative (Rajendran and Sriranjini, 2008). Crithmum maritimum L. (Apiaceae) is a native plant species of Cyprus. A fragrant pickle is prepared from the leaves of this plant with a kind of vinegar named as “girdama” in Cyprus (Viney, 1994). Crithmum maritimum essential oils were previously reported to have considerable antimicrobial (against Bacillus cereus Frankland and Frankland., Staphylococcus aureus Rosenbach., Staphylococcus epidermidis Rosenbach.), antifungal (against Candida albicans (C.P.Robin) Berkhout., C. guillermondii (Syn = Pichia guilliermondii Wick.), Cryptococcus neoformans (San Felice) Vuill., Trichophyton mentagrophytes Priestley., Microsporum canis (Syn = Sabouraudites canis (E. Bodin) Langeron.), Trichophyton rubrum (Castell.) Sabour., Microsporum gypseum (E. Bodin) Guiart & Grigoraki., Epidermophyton floccosum (Harz) Langeron and Miloch., Mycogone perniciosa Mang.), antioxidant (against ABTS radical: 2,2’azinobis3-ethylbenzthiazoline-6-sulphonic acid), antileishmanial (against Leishmania infantum Nicolle.), nematicidal (against Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle.) and insecticidal (against Pheidole pallidula Nylander.) activities (Barbosa et al., 2010; Özcan and Erkmen, 2001; Machado et al., 2010; Tsoukatou et al., 2001; Glamoclija et al., 2009; Marongiu et al., 2007; Houta et al., 2015; Rossi et al., 2007). Essential oil composition of C. maritimum has previously been investigated comprehensively. C. maritumum from different locations of Portugal was reported to contain ␥-terpinene (48.7%), dill apiole (13.8%) in a commercial oil sample (Machado et al., 2010); sabinene (35.3%), ␥-terpinene (29.9%), methyl thymol (18.9%) in a sample from Costa de Caparica (Barroso et al., 1991) and ␥-terpinene (41.5%), sabinene (16.2%), thymol methyl ether (14.9%) in a sample from S. Pedro de Moel, dill apiole (34.0%), ␥-terpinene (27.1%), thymol methyl ether (13.7%) in a sample from Figueira da Foz (Marongiu et al., 2007). Additionally, a seasonal variation study of C. maritimum essential oil from Portugal reports considerable changes in sabinene (7–42%) and ␥-terpinene (26–55%) contents of the oil (Barroso et al., 1992). The studies done by different groups on the samples from different locations of Italy reported the composition of C. maritimum essential oil as ␥-terpinene (37%), methyl thymol (29%), p-cymene (10%) in a sample from Campania (Senatore and De Feo, 1994); as thymol methylether (25.5%), limonene (22.3%), ␥terpinene (22.9%) from Sicily (Ruberto et al., 1991) and as dill apiole (41.0%), ␥-terpinene (29.8%), ␤-phellandrene (13.3%) in a sample from Sardinia (Marongiu et al., 2007). A seasonal variation study of C. maritimum essential oil from Liguria, Italy reported considerable changes in ␥-terpinene (41–68%) composition of the oil (Flamini et al., 1999). Major components of essential oil of Crithmum maritimum from different locations of Turkey were reported as sabinene (26.9%), limonene (24.2%) and ␥-terpinene (19.3%) in a sample from Abant, Bolu (Baser et al., 2000); as methyl thymol (29.8–8.1%), ␥-terpinene (24.5–8.2%), dillapiole (21.5–1.9%), terpinen-4-ol (21.2–2.7%), sabinene (20.5–13.0%), ␤phellandrene (12.8–6.3%) in a sample from Silifke, as ␥-terpinene

(35.2–8.8%), methyl thymol (17.2–7.7%) in a sample from Bodrum (Özcan et al., 2001); as ␤-phellandrene (30.0–13.7%), thymol methylether (24.6–8.7%), (Z)-␤-ocimene (14.3–3.1%), p-cymene (12.8–7.0%), dill apiole (0–20.6%) in samples from Antalya-Mersin, respectively (Senatore et al., 2000); as ␥-terpinene (35.5–32.4%), ␤phellandrene (21.4–22.3%), sabinene (12.6–9.1%) in samples from Sipahili-Yes¸ilovacık Silifke, respectively (Özcan et al., 2006). Until now two well defined chemotypes of C. maritimum were determined from Portugal, one containing high amounts of dill apiol and the other containing very low amount of the same compound (Pateira et al., 1999). In another study variation of dill apiol, sabinene, limonene, ␤-phellandrene, ␥-terpinene, carvacrol methyl ether and thymol methylether content of the essential oil of C. maritimum in different locations of Mediterranean basin (San Sebastian − Spain; Napoli − Italy; Agia Marina, Chios, Crete, Meles − Greece) was reported (Tsoukatou et al., 2001). According to our literature survey we have not encountered any report regarding the insecticidal activity against Sitophilus granarius L., S. oryzae L., Tribolium. castaneum Herbst., T. confusum Jacquelin du Val., Rhyzopertha dominica Fabricius., Oryzaephilus surinamensis L. (Coleoptera), Spodoptera exigua (Hübner) (Lepidoptera) nor the AChE, BChE inhibitory activities of C. maritimum L. essential oil. In the scope of our insecticidal activity screening of the essential oils of edible plants of Cyprus, here we studied the insecticidal activity of C. maritimum from Cyprus. In order to find the possible insecticidal mode of action we have also tested inhibitory effects of the oil on AChE and BChE. Additionally the antimicrobial activity of the essential oil was also tested for potential stored product borne pathogens. Here we present the results of our studies. 2. Materials and methods 2.1. Plant materials Crithmum maritimum was collected on 10th May 2012 from Lapta − Kyrenia coasts. Voucher specimen has been deposited in the Herbarium of the Institute of Environmental Sciences, Near East University, Cyprus (Voucher no. 2550). Plant materials were identified by Dr. Salih Gücel. 2.2. Isolation of the essential oils Leaves (100 g) of the air dried plant was subjected to hydro distillation for 4 h, using a Clevenger- type apparatus to produce essential oils. C. maritimum afforded yellow colored oil from leaves in 0.22% yield (v/w). The oil was preserved in an amber vial under −20 ◦ C until the day it was analyzed. 2.3. Gas chromatography-Mass spectrometry analysis The essential oil analyses were done simultaneously by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS) systems. The GC–MS analyses were done with an Agilent 5975 GC–MSD system with Innowax FSC column (60 m × 0.25 mm, 0.25 ␮m film thickness) and helium was used as a carrier gas (0.8 mL/min). The oven temperature was programmed to 60 ◦ C for 10 min and raised to 220 ◦ C at a rate of 4 ◦ C/min. The temperature was kept constant at 220 ◦ C for 10 min and then raised to 240 ◦ C at a rate of 1 ◦ C/min. The injector temperature was set at 250 ◦ C. Split flow was adjusted at 50:1. Mass spectra were recorded at 70 eV with the mass range m/z 35–450. The GC/MS chromatogram of the leaf oil was given in Fig. 1. The GC analyses were done with Agilent 6890 N GC system. The FID detector temperature was set to 300 ◦ C and same operational conditions applied to a duplicate of the same column used in GC/MS analyses. The simultaneous auto injection was done to obtain the same retention times. The relative

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Fig. 1. GC/MS Chromatogram of Crithmum maritimum leaf oil.

percentage amounts of the separated compounds were calculated from the integration of the peaks in FID chromatograms. Identification of essential oil components was done by comparison of their retention times with authentic samples or by comparison of their relative retention index (RRI) to a series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (McLafferty and Stauffer, 1989; Koenig et al., 2004) and in-house “Baser Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data (Joulain and König, 1998) was used for identification. 2.4. Insect cultures 2.4.1. S. exigua cultures The S. exigua larvae used in the assays were obtained from Cankiri Karatekin University, Yaprakli Vocational School, Laboratory of Animal and Plant Production Department. The insects were reared according to the previously described method (Aydın, 2002). According to this method, 200 larvae obtained from the eggs were transferred to the plastic containers (10 cm × 10 cm; h:9 cm) and fed with the fresh lettuce leaves that changed daily. After 10 days of feeding, groups of 20 larvae were transferred to new plastic containers (20 cm × 15 cm; h:10 cm) and the feeding was continued until the pupa stage is achieved. The adults transformed from the pupa stage were fed on 15% honey solution in water (w/v) in new containers (40 cm × 30 cm; h:10 cm). The eggs of the adults were collected on the oily papers which were placed in the containers. The eggs were treated as explained above for generation of 3rd , 4th and 5th instar larvae which were used in the insecticidal assays. The cultures were developed in a climate chamber at 25 ◦ C and 16:8 h light: dark period. 2.4.2. Stored product insect cultures S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica O. surinamensis were collected from the infested stored products in Turkey. Insect cultures were prepared at Cankiri Karatekin University, Yaprakli Vocational School, in the laboratory of Animal and Plant Production Department according to the previous methods

(Karakoc¸ et al., 2006; Pimentel et al., 2008). According to these methods in order to obtain single aged S. granarius and S. oryzae were introduced in to separate 5 L glass jars which were filled to 1/3 with sterile wheat. Male and female insects were introduced from the stock culture were transferred to the glass jar. After 48 h of incubation, male and female insects were removed from the jars. The infested grains with insect eggs were incubated at 27 ± 2 ◦ C and in a dark climate chamber with 50 ± 10% relative humidity for 45 days. The single aged S. granarius and S. oryzae were used in the assays obtained from this procedure. In order to obtain single aged T. castaneum, T. confusum, O. surinamensis and R. dominica, male and female insects (n = 500) were introduced to separate 1 L glass jars which were filled to 1/3 with a mixture of 70% wheat flour (100 Mesh size) and 30% dry yeast. After 72 h of incubation, male and female insects were removed from the jars. The jars are filled to ½ with the broken wheat which contains insect egg infested flour. The infested grains were incubated at 27 ± 2 ◦ C and 50 ± 10% relative humidity in a dark climate chamber for 45 days and single aged insects were obtained. The single aged T. castaneum, T. confusum, O. surinamensis and R. dominica were used in the assays obtained from this procedure. 2.5. Insecticidal activity assays 2.5.1. Insecticidal contact toxicity assay The C. maritimum essential oil was diluted with acetone to obtain 10% (v/v) stock solution. Stock solution of oil sample (1 ␮L) was applied to the dorsal surface of the thorax of the insects (S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica O. surinamensis) with a 50 ␮L Hamilton syringe (Gökc¸e et al., 2010). The acetone was applied in the blank controls at the same volume. After the sample/blank application the insects (n = 10 for each insect species and treatments) were transferred to the 60 mm glass petri dishes which were filled with 5 g of wheat (for: S. granarius, S. oryzae) or broken wheat (for: T. castaneum, T. confusum, R. dominica O. surinamensis). The insects were incubated at 27 ± 2 ◦ C and 50 ± 10% relative humidity for 72 h. Samples and controls were monitored at 24th h, 48th h and 72th h and the number of dead insects were

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recorded. Each sample and blank treatment was replicated three times. Contact toxicity of C. maritimum essential oil against S. exigua was determined according to the previously described protocol (Karakoc¸ and Gökc¸e, 2012). The essential oil was diluted with acetone to obtain 10% (v/v) stock solution. The essential oil stock solution was applied with 1, 2 and 4 ␮L/larvae amounts to the dorsal surface of the thorax of 3rd , 4th and 5th instar larvae of S. exigua with a 50 ␮L Hamilton syringe, respectively. The acetone was applied in the blank controls at the same volume. After the sample/blank application the insects (n = 10 for each treatment) were transferred to the 60 mm glass petri dishes and fed with fresh lettuce leaves. The insects were incubated at 25 ± 2 ◦ C and 50 ± 10% relative humidity for 24 h. The mortality was recorded after 24 h for each treatment. 2.5.2. Insecticidal fumigant toxicity assay The fumigant toxicity of C. maritimum essential oil against S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica O. surinamensis were determined according to a previous protocol (C¸am et al., 2012). The oil was diluted with acetone to obtain 10% (v/v) stock solution. The 10 mL test tubes were filled with 5 g wheat (for: S. granarius, S. oryzae) or broken wheat (for: T. castaneum, T. confusum, R. dominica O. surinamensis). Adults of S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica, O. surinamensis (n = 10) were introduced in to the separate tubes. The Whatman filter paper N◦ 1 were cut into 1 cm Ø pieces. The 10 ␮L stock solution of the oil is impregnated to the filter pieces with the micropipette as well as the acetone which was used as the blank control. The filter papers were left in open for 5 min in order to evaporate the acetone. The filter papers were attached to the gas tight rubber caps of the test tubes with a needle and the caps were closed. The insects were incubated at 27 ± 2 ◦ C and 50 ± 10% relative humidity for 48 h. Samples and controls were monitored at 48th h. and the number of dead insects were recorded. Each sample and blank treatment was replicated three times. The experiment was designed according to random block pattern. 2.6. Acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activity The inhibitory effects of the C. maritimum essential oil on AChE and BChE were determined with the previously described protocol (Ellman et al., 1961). The assay solution contained 240 ␮L, 1.25 mM 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), 192 ␮L acetylthiocholine iodide (AChI) or butyrylthiocholine iodide (BChI), 1200 ␮L, 100 mM Tris HCl buffer pH = 8.0 and 20 ␮L essential oil solution (10 mg/mL). The blank solution contained 20 ␮L of buffer solution instead of the essential oil. Galanthamine hydrobromide (from Lycoris sp.), was used as a positive control in the assay. Reactions were started by adding 0.0325 U/mL of AChE (Electric Eel) or BChE into the reaction mixture. The reaction was monitored 2 min at the 412 nm wavelength using a spectrophotometer (Carry 60 Single Beam Spectrophotometer, Agilent Technologies, USA). The enzymatic activity was calculated as the percentage of the reaction rate in accordance to the activity obtained from the blank. The data obtained from the linear section of the initial 60 s were used in the calculation of the activities. The AChE, BChE inhibition was calculated by the subtraction of the ratio of the sample activity versus blank activity from the 100. The results of the experiments were given as mean ± standard deviation of three parallel experiments. 2.7. Antimicrobial activity (MIC) assay Three Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Bacillus cereus NRRL B-3711, Bacillus subtilis NRRL B-4378) and two Gram-negative bacteria (Escherichia coli NRRL B-3008, Salmonella

typhimurium ATCC 13311) were used in this study. The minimum inhibitory concentration (MIC) values were determined for the C. maritimum oil, on each organism by using microplate dilution method (Iscan et al., 2002). Stock solution of the oil (2 mg/mL) and standart antibacterial compounds ampisilin and clarithromycin (2 mg/mL) were prepared with sterile DMSO. Liquid medium was diluted by adding 25% DMSO. Serial dilution was done on 96-well microplates. Bacteria were standardized according to McFarland No:0.5 after incubation for 24 h at 37◦ C on MHB (CLSI, M7-A7, 2006). Cultures were mixed with the essential oil and were incubated for 24 h at 37◦ C. Minimum inhibitory concentrations (MIC: ␮g/mL) were detected at the minimum concentration where bacterial growth was missing. 1% 2,3,5-Triphenyltetrazolium chloride (TTC, Aldrich St. Louis MO, USA) was used as an indicator of bacterial growth. Essential oil free solutions were used as negative control and ampisilin, clarithromycin were used as the positive controls. All of the experiments were performed in triplicate and means of results were given for the MIC values of the oils.

2.8. Statistical analysis The activity results obtained in the insecticidal activity assays were transformed in to percent death values; which were used to obtain the arcsin values (Zar, 1996). The arcsin values were subjected to variance analysis (ANOVA) followed by Tukey’s multiple comparison test with P < 0.05 significance level. All of the statistical analysis were performed with Minitab Release 14 (Mckenzie and Goldman, 2005) program.

3. Results The essential oil composition of Crithmum maritimum was given in Table 1. Twenty compounds were detected representing 100.0% of C. maritimum leaf oil. The essential oil was dominated by the monoterpenes. The major components of the essential oil were ␥terpinene (39.3%), ␤-phellandrene (22.6%), carvacrol methyl ether (10.4%), (Z)-␤-Ocimene (8.2%) and p-cymene (6.4%). The insecticidal contact toxicity of the C. maritimum oil against S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica and O. surinamensis are given in Table 2. C. maritimum essential oil was also investigated for its contact toxicity against S. exigua at different development stages (3rd , 4th and 5th instar larvae). The highest toxicity observed against S. exigua was for 3rd instar larvae which caused 89% mortality. The oil caused 50% mortality on 4th instar larvae and showed no activity in the 5th instar larvae at the studied concentrations. The highest contact toxicity of the oil was against S. oryzae which produced 46.65%, 83.64% and 93.30% mortality after 24 h, 48 h, 72 h of the application, respectively. The oil also had considerable contact toxicity against R. dominica and O. surinamensis which caused 83.26% and 70.33% mortality after 72 h of the application, respectively. Essential oil produced medium insecticidal activity on S. granarius (50.00% after 72 h) and low insecticidal activity on T. castaneum (22.16% after 72 h) and T. confusum (33.26% after 72 h). The essential oil afforded significantly higher activity than the negative control. The insecticidal fumigant toxicity of the C. maritimum oil against S. granarius, S. oryzae, T. castaneum, T. confusum, R. dominica and O. surinamensis were given in Table 3. Highest fumigant toxicity was observed on S. granarius and S. oryzae which was 100% mortality after 48 h of application. O. surinamensis was also highly susceptible to the oil in the fumigant application. A high mortality 90.75% was observed after 48 h of application for O. surinamensis. The essential oil did not produced any mortality on T. castaneum, T. confusum and very low mortality (4.53%) on R. dominica in fumigant application.

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Table 1 Essential oil composition of Crithmum maritimum L. leaves. No

RRI

Compound

%

Identification Method

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1032 1035 1118 1132 1174 1176 1188 1203 1218 1246 1255 1266 1280 1290 1604 1611 1614 1706 1726 2186

␣-Pinene ␣-Thujene ␤-Pinene Sabinene Myrcene ␣-Phellandrene ␣-Terpinene Limonene ␤-Phellandrene (Z)-␤-Ocimene ␥-Terpinene (E)-␤-Ocimene p-Cymene Terpinolene Thymol methyl ether (=methyl thymol) Terpinen-4-ol Carvacrol methyl ether (=methyl carvacrol) ␣-Terpineol Germacrene D Eugenol Total

2.3 0.5 0.2 4.4 1.0 1.0 0.2 1.0 22.6 8.2 39.3 1.0 6.4 tr 0.2 0.6 10.4 0.2 tr 0.5 100.0

a,b a a,b a,b a,b a,b a,b a,b a,b a a,b a a,b a,b a,b a,b a,b a,b a a,b

RRI: Relative retention indices calculated against n-alkanes, % calculated from FID data; tr: Trace (< 0.1%); Identification Method; a: comparison of mass spectra with the Wiley and Mass Finder libraries and retention times; b: comparison with genuine compounds on the HP Innowax column.

Table 2 Insecticidal Contact Toxicity of Crithmum maritimum oil. 24 ha Insect species Sitophilus granarius Sitophilus oryzae Tribolium castaneum Tribolium confusum Rhyzopertha dominica Oryzaephilus surinamensis

48 h

b

c

Oil 10.00 ± 0.00 cd 46.65 ± 0.19 ab 19.31 ± 0.96 c 26.20 ± 0.94 bc 67.09 ± 0.94 a 56.84 ± 0.81 a

Control 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e

72 h

Oil 23.18 ± 0.26 d 83.64 ± 0.35 a 19.31 ± 0.96 d 33.26 ± 0.21 cd 67.09 ± 0.94 ab 56.84 ± 0.81 bc

Control 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 4.53 ± 1.97 e 4.53 ± 1.97 e

Oil 50.00 ± 0.58 abc 93.30 ± 3.20 a 22.16 ± 1.91 c 33.26 ± 0.21 bc 83.26 ± 7.50 ab 70.33 ± 0.70 abc

Control 0.00 ± 0.00 e 0.00 ± 0.00 e 4.53 ± 1.97 e 4.53 ± 1.97 e 4.53 ± 1.97 e 4.53 ± 1.97 e

a

The contact insecticidal activity results were given at three different time intervals 24, 48 and 72 h. The results were given as the percent mortality of the insects given as mean of three parallel experiments that was repated three times ± standard error. c The negative control is sample free acetone. d Different letters after the standard error values represent statistical significance of these values when compared with the results of other insect species (ANOVA, P < 0.05, Tukey’s Test). e Indicates the insecticidal activity result of the substances is significantly different than the negative control. b

Table 3 Insecticidal Fumigant Toxicity of Crithmum maritimum oil.

Table 4 Antimicrobial (MIC) activity of the Crithmum maritimum essential oil.

Insect species

Critimum maritimuma

Controlb

Microorganism

Oil

Ampisilin

Clarithromycin

Sitophilus granarius Sitophilus oryzae Tribolium castaneum Tribolium confusum Rhyzopertha dominica Oryzaephilus surinamensis

100.00 ± 0.00 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.00 ± 0.00 b 4.53 ± 3.41 b 90.75 ± 7.00 a

0.00 ± 0.00e 0.00 ± 0.00e 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00e

Escherichia coli NRRL B-3008 Staphylococcus aureus ATCC 6538 Salmonella typhimurium ATCC 13311 Bacillus cereus NRRL B-3711 Bacillus subtilis NRRL B-4378

1250 1250 1250 1250 1250

4 1 1 0.06 0.06

64 2 2 0.125 0.125

c

d

MIC results were given as the ␮g/mL and as the mean of three parallel experiments.

a

The contact insecticidal activity results were given at three different time intervals 24, 48 and 72 h. b The results were given as the percent mortality of the insects given as mean of three parallel experiments that was repated three times ± standard error. c The negative control is sample free acetone. d Different letters after the standard error values represent statistical significance of these values when compared with the results of other insect species (ANOVA, P < 0.05, Tukey’s Test). e Indicates the insecticidal activity result of the substances is significantly different than the negative control.

The results of antimicrobial activity tests against the selected microorganisms were given in Table 4. The C. maritimum essential oil showed very low activity against all of the tested microorganisms when compared to the positive controls. The antimicrobial activity is as low as 10000 fold smaller than the positive controls for B. cereus and B. subtilis. 4. Discussion

The AChE & BChE inhibitory activity of the oil was determined at 121 ␮g/mL final concentration of the oil in the assay mixture. The oil produced 50.3% inhibition of the AChE and 59.8% inhibition of BChE. The activity of the oil corresponds to the activity of 0.766 ␮g/mL and 2.764 ␮g/mL (assay concentrations) of galanthamine for AChE and BChE assays respectively.

The C. maritimum essential oil produced high contact activity against S. oryzae. However the activity observed for S. granarius was significantly lower. S. oryzae was more susceptible to the essential oil in contact toxicity test. Previously fumigant insecticidal activity of essential oil that is rich in ␥-terpinene and pure ␥-terpinene against S. oryzae was reported (Kedia et al., 2015). Another report

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indicates the high activity of essential oils from Zanthoxylum sp. against S. oryzae which contains high amounts of ␤-myrcene and ␤-phellandrene (Prieto et al., 2011). The oil produced very high fumigant toxicity against both Sitophilus species tested similar to the previous reports. The ␥-terpinene and ␤-phellandrene content of the essential oil could be responsible for the observed activity against S. oryzae. The oil also produced considerable activity against R. dominica and O. surinamensis in contact toxicity assay. However, at the same concentration and time interval activity observed for S. oryzae was significantly higher. The R. dominica was more susceptible to the essential oil than the O. surinamensis in the contact toxicity assay. However, the essential oil did not show any activity in the fumigant insecticidal activity assay against R. dominica but showed very high activity against O. surinamensis. This could suggest that the essential oil have a different mode of action on R. dominica than the other tested insects. A similar situation was also observed for S. granarius. The oil showed a medium activity against S. granarius in the contact insecticidal activity assay. However, very high fumigant insecticidal activity was observed against the same insect. The essential oil has very low insecticidal activity against Tribolium species in both fumigant and contact toxicity assays. The oil also showed considerable activity against S. exigua 3rd instar larvae but was not active in other development stages. Interestingly, the oil also produced a higher inhibitory effect on BChE when compared with its AChE inhibition at the same concentration. Previously, AChE inhibition of a selection of monoterpenoids including ␥-terpinene was investigated and these substances were reported as weak inhibitors. However, in the same report AChE enzyme (from electric eel) was reported to be capable of lodging more than one monoterpene as inhibitor (López et al., 2015). Additionally, the AChE inhibitory property of the second major component of the oil ␤-phellandrene was reported to have moderate inhibitory effect (Seo et al., 2014). The observed inhibitory effects of the C. maritimum essential oil on AChE and BChE are probably due to the collective effect of all the components of the oil. The oil was also tested against a selection of pathogens that could be found on stored products. The oil produced very low antimicrobial activity against all of the microorganisms tested. Highest antimicrobial activity of the oil was observed at 300 fold higher minimum inhibitory concentration than the positive control (Ampisilin) against Escherichia coli.

5. Conclusion The essential oil of C. maritimum from Cyprus afforded high fumigant and contact insecticidal activity against selectively to the major stored product insects namely S. oryzae and O. surinamensis. Due to the edible uses of this plant species the essential oil could also be regarded as safe. Therefore C. maritimum essential oil could be a promising fumigant agent against the indicated stored product pests. However studies regarding its application, interaction with the stored product as well as its toxicological assessments are still required.

Acknowledgements The results presented in this article was obtained using the infrastructure provided by the TUBI˙ TAK − The Scientific and Technological Research Council of Turkey (Project No: TOVAG 111O138) and BAPK project of I˙ stanbul Kemerburgaz University (Project No: PB-011-2013). The results given in this article are partially presented in 45th International Symposium on Essential Oils.

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