Comparative and synergistic activity of Rosmarinus officinalis L. essential oil constituents against the larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni (Lep., Noctuidae) Jun-Hyung Tak, Eduardo Jovel, and Murray B. Isman* Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada BACKGROUND: Plant essential oils are usually complex mixtures, and many factors can affect their chemical composition. To identify relationships between composition and bioactivity of the constituents, comparative and synergistic interactions of the major constituents of rosemary essential oil were evaluated against third instar larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni via different methods of application. RESULTS: The major constituents of the rosemary oil we used were 1,8-cineole, (±)camphor, (+)-α-pinene and camphene. Via topical application to larvae, 1,8-cineole was identified as the major active compound, whereas via fumigation, 1,8-cineole and (±)camphor, and in a cytotoxicity assay, (+)-α-pinene, were determined to be the major active principles. Several combinations of these constituents exhibited synergistic insecticidal activities when topically applied, particularly among combinations of three major constituents, (±)-camphor, (+)-α-pinene, and camphene. A binary mixture of 1,8-cineole and (±)-camphor showed enhanced activity, with a synergy ratio of 1.72. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ps.4010
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CONCLUSION: Based on our results, the insecticidal activity of rosemary oil appears to be a consequence of the synergistic interaction between 1,8-cineole and (±)-camphor, and (±)camphor should be considered a promising synergizing agent.
KEYWORDS: 1,8-cineole; (±)-camphor; synergy; toxicity; botanical insecticide
1 INTRODUCTION General concern about the risks associated with the repeated use of synthetic insecticides has led to searching for more environmentally benign and sustainable tools for pest control, as evidenced by the growth of organic produce in supermarkets.1 Botanical insecticides have long been touted as attractive alternatives to conventional pesticides because many botanicals pose little threat to the environment or to human health.2 The bioactivity of rosemary (Rosmarinus officinalis L.) essential oil has been reported for many insect and acarine pest species,3-7 and microorganisms including several food-borne pathogenic species of bacteria and fungi.8-9 Plant essential oils are complex mixtures of low molecular weight terpenoid compounds, and their chemical compositions can vary based not only on biological factors including genotype and interactions with other organisms, but also environmental factors such as climate, altitude, and nutrition levels. Chemical variation in rosemary essential oils has been well documented,10-12 and since these variations can influence biological activities, especially insecticidal activity,13 understanding the contribution of individual constituents of an essential oil as well as their interactions with respect to insecticidal activity is important for ‘quality control’ of a commercialized biopesticide based on this essential oil. In the present study, our This article is protected by copyright. All rights reserved
objective was to identify the contributions of individual essential oil constituents via different application methods, and assess any synergistic or antagonistic interactions in terms of insecticidal activity.
2 MATERIALS AND METHODS 2.1 Chemicals Rosemary essential oil (Intarome Fragrance & Flavors Corp. Norwood, NJ, USA) was obtained from EcoSafe Natural Product Inc. (Saanichton, BC, Canada). Standard compounds of constituents of the oil were purchased from Sigma-Aldrich (camphene [95%], (±)-camphor [96%], 1,8-cineole [99%], (R)-(+)-limonene [97%], (+)-α-pinene [98%], and (-)-β-pinene [99%], St Louis, MO, USA), and Thermo Fisher Scientific ((-)-borneol [97%], L-bornyl acetate [97%], p-cymene [≥99%] and α-terpineol [98%], Waltham, MA, USA). AlamarBlue® dye, Express Five® SFM medium, gentamicin and L-glutamine were purchased from Life Technologies (Carlsbad, CA, USA).
2.2 Gas chromatography-mass spectrophotometry Major constituents of rosemary oil were analyzed by the static headspace method on an Agilent 7890/5975C gas chromatograph inert XL mass spectrometer (Agilent Technologies Canada Inc., Ottawa, ON, Canada) operating in electron ionization mode fitted with a J&W DB-WAX 30 m × 0.25mm ID, 0.25 μm thickness fused silica column. The injection was done in a split mode (50:1) with the volume of 250 μL. The oven temperature setup was programed as 40 ºC for 1 min, followed by an increase of 10 ºC min-1 to 85 ºC, and then 35
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ºC min-1 to 250 ºC and held for 4 min with the total run time of 14.2 min. Helium was used as a carrier with 0.9 mL min-1 of flow. The data were analyzed using Enhanced Chemstation software and the constituents were identified by matching the spectra against the Wiley09/Nist08 MS library. The analysis was confirmed by re-analyzing an artificial essential oil, prepared by mixing the authentic standard compounds (> 1% in concentration) in their proportions found in the natural oil, under the same conditions. 2.3 Insect and cell line maintenance 2.3.1 Insect maintenance Eggs of the cabbage looper, Trichoplusia ni were obtained from the Great Lakes Forestry Centre, Natural Resources Canada (Sault Ste. Marie, ON, Canada). The colony was reared on pinto bean based artificial diet (Bio-Serv Inc., Frenchtown, NJ, USA) in the insectary at the University of British Columbia, Vancouver, BC, at 22-25 ºC and a 16:8 h LD photoperiod. 2.3.2 Cell line maintenance An ovarian cell line of T. ni (High Fivetm, Life Technologies) was grown in Express Five® SFM medium, supplemented with 1 mM L-glutamine and 50 μg mL-1 gentamicin. For long term maintenance of the cell line, subculture was performed twice a week with a split ratio of 1:11 to 2:10 (cell suspension : new medium) and cells were allowed to grow at 27 ºC in an incubation chamber.
2.4 Insecticidal and cytotoxic activity of essential oil and individual constituents 2.4.1 In vivo insecticidal activity A topical application method using third-instar larvae of the cabbage looper was conducted This article is protected by copyright. All rights reserved
using a Hamilton Microliter syringe attached to a repeating dispenser. Each larva received 1 μL of acetonic solution of the test compounds or acetone alone as the control. Three replications of ten treated larvae were each transferred into a 7 cm dia Petri dish with 0.5 g of artificial diet, and kept under the same conditions as for insect maintenance. After 24 h, mortality was recorded; larvae were determined to be dead when no movement was observed by probing the insect with a fine brush. Four to six different concentration levels were used to estimate LD50 values. 2.4.2 In vitro cytotoxicity T. ni cells were collected 72 h after the subculture and diluted to a density of 2 × 105 cells mL-1 with fresh medium. Fifty μL of cell suspension was loaded into a 96-well plate (Thermo Fisher Scientific), and allowed to settle for 2 h in the incubation chamber. Target compounds were dissolved in sterilized dimethyl sulfoxide (DMSO) and filtered with a nylon syringe filter with a pore size of 0.2 μm (Thermo Fisher Scientific), and then the filtered solution was mixed with fresh medium (1:149, v/v). Fifty μL of treatment solution was applied to each well of the plate, with DMSO/medium solution alone applied to control cells. After 48 h of incubation at 27 ºC, cells were inspected under Axiovert 35 inverted florescence microscope (Zeiss, Oberkochen, Germany) and viability of cells was monitored by a fluorescent method. Ten μL of alamarBlue® dye was added into each well, and fluorescence intensity was measured after 15 min of incubation using a Polarstar galaxy spectrophotometer (544 nm excitation and 590 nm emission, BMG Labtechnologies, Ortenberg, Germany). Nine concentrations with three replications were used to determine the IC50 values of essential oil and constituents. The intensity of the fluorescence was directly proportional to the number of metabolically active cells, and inversely proportional to the toxicity of the tested samples (Figure 1). This article is protected by copyright. All rights reserved
2.5 Comparative activities via different application methods To evaluate the individual constituents’ contribution to overall activity, a compound elimination assay was conducted using a method similar to that previously described. 7 A series of artificial essential oils was prepared with the constituents in their natural proportions, either as the full mixture or with one constituent missing. Each artificial oil had the same amounts of individual compounds as in the full mixture oil, with the amount of missing compound replaced by acetone. Two dose or concentration levels (LD/LC95 and LD/LC50) of each artificial oil were applied topically or by a fumigation method, and double the IC50 value was applied in the cytotoxicity assay. To determine the LC95 and LC50 values of rosemary essential oil by fumigation, ten third instar larvae were transferred into a 5.5 cm dia Petri dish with 0.5 g of artificial diet on a filter paper (4.25 cm diameter, Thermo Fisher Scientific) and covered with a ventilated lid (70 holes created by a heated syringe), and the edge was sealed with Parafilm (insect chamber). It was assumed that the larvae were exposed to the entire volatilized constituents of rosemary oil since the volume of the chamber was relatively small (25.3 mL). Six different concentrations of 50 μL of an acetonic solution of the essential oil were applied onto another filter paper. After 30 sec, the filter paper was attached to the bottom of a new Petri dish using adhesive tape (fumigant chamber), and both chambers were combined and sealed with Parafilm. Since the diameter of holes was smaller than that of the larvae, and there was a gap between the treated filter paper and the inner lid, larvae were prevented from having direct contact with the treated filter paper, but were exposed to the evaporated compounds. Mortality was determined after 24 h, and the compound elimination assay via fumigation was conducted in the same fashion. All tests were repeated three times. This article is protected by copyright. All rights reserved
2.6 Synergistic interactions among four major constituents To evaluate potential synergies between the four major constituents (1,8-cineole, (±)-camphor, (+)-α-pinene and camphene), mixtures were prepared following either the actual constituent ratio based on chemical analysis of the oil, or a 1:1 ratio of the compounds. Mixtures were applied topically to 3rd instar larvae, and their LD50 values were estimated after 24 h. To determine the relationships of the mixtures, we used two statistical models to compare expected and observed LD50 values, Hewlett and Plackett’s model14 and Wadley’s model.15 Based on Hewlett and Plackett’s calculation, the expected LD50 values (assuming additive interaction) were determined from the equation =
×
( )
+
×
( )
+
×
( )
+ ⋯ +
×
( )
where a was the proportion of compound A in the mixture, and LD50(a) was the LD50 of compound A. According to Wadley, theoretical LD50 values were calculated from + + + ⋅⋅⋅ +
= ( )
+
( )
+
( )
+ ⋅⋅⋅ +
( )
The interaction between the observed and theoretical LD50 values were compared as
=
expected LD observed LD
The relationship between the constituents of the mixture was defined as either synergistic (when R > 1.5), additive (1.5 ≥ R > 0.5) or antagonistic (R ≤ 0.5) based on this model.
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2.7 Data analysis Mortality and cytotoxicity data from the compound elimination assay were subjected to analysis of variance (ANOVA), and probit analysis was used to calculate the LD/LC95 and LD/LC50 values using StatPlus 2009 (version 5.8.4, AnalystSoft, Alexandria, VA, USA). IC50 values were determined by using GraphPad Prism 5 (Version 5.02, GraphPad Software Inc., La Jolla, CA, USA).
3 RESULTS 3.1 Chemical composition of rosemary essential oil Based on the GC-MS analysis of rosemary essential oil constituents, 1,8-cineole (37.6%) was the most abundant compound followed by (±)-camphor, (+)-α-pinene, and camphene (Table 1). These four major constituents comprised more than 80% of the total weight of the oil and all of the remaining individual constituents comprised less than 5% of the concentration, with a total of 97.7% of the constituents identified.
3.2 Insecticidal activity and cytotoxicity of individual constituent In third instar larvae of the cabbage looper, the LD50 value of rosemary oil was 215.8 μg insect-1 (Table 2). Among the constituents tested, α-terpineol was the most potent (61.9 μg insect-1), whereas camphene was inactive at the highest concentration treated (> 1,000 μg insect-1). Although 1,8-cineole showed activity comparable to the complete oil, none of the major compounds (> 5% of the oil) had LD50 values lower than that of the intact oil. This article is protected by copyright. All rights reserved
In the in vitro cytotoxicity assay, the most active compound was (R)-(+)-limonene (49.8 μg mL-1), and several constituents including 1,8-cineole (> 1,000) and (±)-camphor (> 100) failed to show a cytotoxic effect on even at the highest concentrations tested.
3.3 Contribution of individual compound to overall activity At the higher concentration of topical application (LD95 of rosemary oil), there was a significant decrease in mortality when 1,8-cineole was eliminated from the artificial oil (Figure 2A). Although mortality decreased slightly when (±)-camphor was excluded, it was not statistically different (P < 0.05). In the fumigation assay against 3rd instar larvae of T. ni, rosemary essential oil gave 175.8 and 107.4 μg mL-1 air as LC95 and LC50 values, respectively. Based on these concentrations, the compound elimination assay revealed that 1,8-cineole and (±)-camphor were significantly more active than all the other constituents (P < 0.05, Figure 2B). In the cytotoxicity assay, the most significant decrease of activity occurred when (+)-αpinene was eliminated, but the two major constituents, 1,8-cineole and (±)-camphor did not decrease the cytotoxic effect when they were missing (Figure 2C).
3.4 Interactions among mixtures Insecticidal synergistic interactions among the four major constituents (1,8-cineole, (±)camphor, (+)-α-pinene and camphene) were investigated (Table 3). Combinations following natural proportions of the essential oil composition showed several synergistic relationships; the most significant synergy based on Wadley’s determination was the mixture of (±)This article is protected by copyright. All rights reserved
camphor+(+)-α-pinene+camphene (R = 3.82). A binary mixture of the two most abundant constituents, 1,8-cineole+(±)-camphor, produced a significantly lower LD50 than for the individual compounds. A notable trend of synergy was found with (±)-camphor. Apart from the full mixture of the four compounds, all other combinations that included (±)-camphor as an element showed synergy (R > 1.5). When the constituents were mixed at 1:1 ratio, (±)-camphor+camphene showed the greatest increase in toxicity (R = 2.28), followed by (±)-camphor+(+)-α-pinene and (±)-camphor+(+)α-pinene+camphene. No combination tested showed an antagonistic interaction.
4 DISCUSSION Our compound elimination bioassays pointed to 1,8-cineole as the major insecticidal constituent of rosemary oil based on topical administration, and 1,8-cineole and (±)-camphor as major insecticidal constituents via fumigation to the cabbage looper. Although α-terpineol had the lowest LD50 value when applied topically, its concentration in the oil is relatively small (1.7%), and elimination of it from the artificial mixture did not lead to a decrease in mortality, suggesting that its contribution to overall toxicity is limited. In a previous study of our laboratory of the toxicity of rosemary oil to the two-spotted spider mite, Tetranychus urticae,7 1,8-cineole showed the greatest contribution to overall activity, followed by (+)-αpinene. In another study of basil essential oil (Ocimum kilimandscharicum), the major constituent, (±)-camphor, exhibited not only high insecticidal activity but was also significant repellent and inhibited growth and development of four species of stored product beetles.16 It is often observed that crude essential oils are considerably more active than any of their individual constituents.17-19 Our results clearly indicated several synergistic interactions This article is protected by copyright. All rights reserved
between constituents of rosemary oil; all combinations of the major constituents tested produced toxicity comparable to or greater than that expected. The two major constituents, 1,8-cineole and (±)-camphor are synergistic (LD50 = 155.6 μg insect-1, R = 1.72). Taking into consideration concentrations of these constituents in the essential oil (57.8% combined) and their enhanced toxicity when combined, the insecticidal activity of rosemary oil might be mainly a consequence of their synergistic interaction. Since plant essential oils obtained from ‘actual’ plants vary chemically based on numerous biotic and abiotic factors, the chemical profile of oils will not be uniform, except where oils from different sources are intentionally blended to achieve a consistent composition. We previously reported variation in response of noctuid larvae based on variation in chemical composition of rosemary oil,13 with LD50 values ranging from 58.9 to 335.9 μg insect-1 in T. ni, and 167.1 to 372.1 μg insect-1 in the armyworm, Pseudaletia unipuncta. This points to the importance of understanding the contributions of individual constituents and combinations thereof to overall insecticidal activity as a basis for standardization and quality control of commercial insecticides based on plant essential oils. Interestingly, among the four major constituents of rosemary oil, (±)-camphor displayed the most consistent synergistic effects with other compounds. Eleven out of 14 combinations that included (±)-camphor showed synergy, and the remaining 3 combinations showed at least 20% increases in insecticidal activities (Table 3). In a similar study of synergy among 30 insecticidal compounds to Spodoptera littoralis, (±)-camphor exhibited synergy with 22 substances, and 9 of the mixtures with (±)-camphor showed the maximal synergic effect.20 It is still unclear how this synergy is achieved, but based on pharmacological studies Wagner and Ulrich-Merzenich suggested four possible mechanisms; (a) synergistic multi-target effects, (b) pharmacokinetic or physicochemical effects based on improved solubility and
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absorption rate, (c) interactions of agents with resistance mechanisms, and (d) respective elimination or neutralization of adverse effects.21 Since plant essential oils are usually complex mixture of compounds, they can be expected to have multiple modes of action. For example, 1,8-cineole has been reported to have inhibitory activity on AChE, associated with octopamine
23
and GABA receptors.
24
22
but it is also
Also, some essential oils have been
shown to inhibit detoxifying enzymes, resulting in synergy with synthetic insecticides.
25
To
date, increased cuticular penetration by essential oil constituents as a mechanism of synergy has not been reported, but increased penetration by enhanced solubility might also be involved as the constituents often have a wide range of hydrophilic-hydrophobic properties. Synergistic insecticidal activities have been observed in many cases, not only between constituents of essential oils, but also between essential oils, synthetic insecticides,
28
26
monoterpene compounds,
and between essential oils and synthetic compounds.
29
27
Several
statistical models have been proposed to determine synergy, and in the present study we adopted two models, Hewlett and Plackett’s calculation and Wadley’s. Some of the estimated LD50 values were similar, but in general, Wadley’s model was deemed to be the more conservative of the two. One shortcoming of these two models is the time and effort required to obtain the empirical LD50 values of the individual compounds as well as those of mixtures. A somewhat more convenient model is Finney’s calculation.
30
Expected mortality of the
mixture can be calculated using the equation: E = Oa + Ob (1 – Oa), where E is the expected mortality, Oa and Ob are the observed mortalities of the individual compounds. A chi-square value is to be obtained via: χ2 = {(Om – E)2} / E, where Om is observed mortality of the mixture. The calculated chi-square can be compared to a chi-square tabular value with df = 1 and α = 0.05, which gives 3.84. If the chi-square is > 3.84, synergy is confirmed. However, caution must be used with this approach. Being based on single concentrations or doses, this
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method is considerably less precise than models based on LD50 values, and depends on appropriate selection of the single doses used. We found that neither 1,8-cineole or (±)-camphor nor their mixture had a potent cytotoxic effect on the T. ni cell line (IC50 > 1,000 μg mL-1, > 100 μg mL-1, > 1,000 μg mL-1, respectively). One of the possible reasons for this could be the lower solubility of the compounds in the cytotoxicity assay system, because we used a relatively low concentration of DMSO as a carrier, and no surfactant. While it is possible that some of the test compounds were not fully in solution at the higher concentrations tested, we believe that at lesser concentrations the compounds would have been completely solubilized. Insect cell systems have been investigated as a convenient tool to screen for insecticidal bioactivity, as a replacement for bioassays with live insects. However, the rapid insecticidal action of plant essential oils to some insect species suggests one or more neurotoxic modes of action. 2 It has been reported that certain biochemical targets such as octopamine 31 or GABA 32
receptors are associated with the insecticidal mode of action of monoterpene compounds.
Decombel et al. 33 reported weak correspondence between in vivo insecticidal activity and in vitro cytotoxicity of synthetic neurotoxic insecticides. Moreover, no direct association (high mortality to mites / high cytotoxicity to an insect cell line) was found in a screening study of 67 plant extracts. 34 Cell culture systems may be useful in identifying prospective insecticides with modes-of-action based on disruption of primary metabolism, protein synthesis and other activities common to most animal cells, but not for substances that depend on receptors found only in the nervous system (i.e., those with a neurotoxic mode of action). It has been more than 15 years since essential oil-based pesticides were introduced into the US market. EcoSMART Technologies is the leading company specialized in this area, having introduced a range of agricultural, professional and consumer botanical insecticides using This article is protected by copyright. All rights reserved
rosemary oil and other essential oils derived from clove leaf (Syzygium aromaticum), peppermint (Mentha × piperita), cinnamon leaf (Cinnamomum zeylandicum), lemongrass (Cymbopogon nardus) and thyme (Thymus vulgaris) as active ingredients.
35
Understanding
synergistic mechanisms within and between essential oils and their constituents may facilitate their further commercial development toward more favorable pest management products.
ACKNOWLEDGMENTS The authors gratefully acknowledge Nancy Brad, Zyta Abramowski and Lina Madilao for technical supports. This work was supported by a University of British Columbia graduate fellowship [to JHT] and a Discovery grant from the Natural Sciences and Engineering Research Council of Canada [2729-11] [to MBI].
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9 Bozin B, Mimica-Dukic N, Samojlik I, and Jovin E, Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae) essential oils. J Agric Food Chem 55:7879-7885 (2007). 10 Elamrani A, Zrira S, Benjilali B and Berrada M, A study of Moroccan rosemary oils. J Essent Oil Res 12:487-495 (2000). 11 Serrano E, Palma J, Tinoco T, Venâncio F and Martins A, Evaluation of the essential oils of rosemary (Rosmarinus officinalis L.) from different zones of “Alentejo” (Portugal). J Essent Oil Res 14:87-92 (2002). 12 Salido S, Altarejos J, Nogueras M, Saánchez A and Luque P, Chemical composition and seasonal variations of rosemary oil from Southern Spain. J Essent Oil Res 15:10-14 (2003). 13 Isman MB, Wilson JA and Bradbury R, Insecticidal activities of commercial rosemary oils (Rosmarinus officinalis) against larvae of Pseudaletia unipuncta and Trichoplusia ni in relation to their chemical compositions. Pharm Biol 46:82-87 (2008). 14 Don-Pedro KN, Investigation of single and joint fumigant insecticidal action of citruspeel oil components. Pestic Sci 46:79-84 (1996). 15 Gisi U, Binder H and Rimbach E, Synergistic interactions of fungicides with different modes of action. Trans Br mycol Soc 85:299-306 (1985). 16 Obeng-Ofori D, Reichmuth CH, Bekele AJ and Hassanali A, Toxicity and protectant potential of camphor, a major component of essential oil of Ocimum kilimandscharicum, against four store product beetles. Int J Pest Manage 44:203-209 (1998). 17 Hummelbrunner LA and Isman MB, Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., This article is protected by copyright. All rights reserved
Noctuidae). J Agric Food Chem 49:715-720 (2001). 18 Akhtar Y, Pages E, Stevens A, Bradbury R Camara CAG and Isman MB, Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper. Physiol Entomol 37:81-91 (2012). 19 Jiang Z, Akhtar Y, Bradbury R, Zhang X and Isman MB, Comparative toxicity of essential oils of Litsea pungens and Litsea cubeba and blends of their major constituents against the cabbage looper, Trichoplusia ni. J Agric Food Chem 57:4833-4837 (2009). 20 Pavela R, Acute, synergistic and antagonistic effects of some aromatic compounds on the Spodoptera littoralis Boism. (Lep., Noctuidae) larvae. Ind Crops Prod 60:247-258 (2014). 21 Wagner H and Ulrich-Merzenich, Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine 16:97-110 (2009). 22 Abdelgaleil SAM, Mohamed MIE, Badawy MEI and El-arami SAA, Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase activity. J Chem Ecol 35:518-525 (2009). 23 Zhukovskaya MI, Aminergic regulation of pheromone sensillae in the cockroach Periplaneta americana. J Evol Biochem Physiol 43:318-326 (2007). 24 Tong F and Coats JR, Quantitative structure–activity relationships of monoterpenoid binding activities to the housefly GABA receptor. Pest Manag Sci 68:1122-1129 (2012). 25 Tong F and Bloomquist JR, Plant essential oils affect the toxicities of carbaryl and permethrin against Aedes aegypti (Diptera: Culicidae). J Med Entomol 50:826-832 (2013). 26 Ngamo TSL, Ngatanko I, Ngassou MB, Mapongmestsem PM and Hance T, Insecticidal efficiency of essential oils of 5 aromatic plants tested both alone and in combination towards This article is protected by copyright. All rights reserved
Sitophilus oryzae (L.) (Coleoptera : Curculionidae). Research Journal of Biological Sciences 2:75-80 (2007). 27 Pavela R, Acute and synergistic effects of monoterpenoid essential oil compounds on the larvae of Spodoptera littoralis. J Biopestic 3:573-578 (2010). 28 Pennetier C, Corbel V and Hougard JM, Combination of a non-pyrethroid insecticide and a repellent: a new approach for controlling knockdown-resistant mosquitoes. Am J Trop Med Hyg 72:739-744 (2005). 29 Shaalan EAS, Canyon D, Younes MWF, Abdel-Wahab H and Mansour AH, A review of botanical phytochemicals with mosquitocidal potential. Environ Int 31:1149-1166 (2005). 30 Trisyono A and Whalon ME, Toxicity of neem applied alone and in combinations with Bacillus thuringiensis to Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 92:1281-1288 (1999). 31 Enan EE. Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to essential oils. Arch Insect Biochem Physiol 59:161-171 (2005). 32 Tong F and Coats JR, Effects of monoterpenoid insecticides on [3H]-TBOB binding in house fly GABA receptor and 36Cl− uptake in American cockroach ventral nerve cord. Pestic Biochem Physiol 98:317-324 (2010). 33 Decombel L, Smagghe G and Tirry L, Action of major insecticide groups on insect cell lines of the beet armyworm, Spodoptera exigua, compared with larvicidal toxicity. In Vitro Cell Dev Biol-Animal 40:43-51 (2004).
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Table 1. Chemical constituents of rosemary essential oil no.
constituent
retention time (min)
% area
1
(+)-α-pinene
4.19
15.7
2
α-fenchene
4.62
0.2
3
camphene
4.72
7.9
4
(-)-β-pinene
5.22
4.3
5
(R)-(+)-limonene
6.29
2.1
6
1,8-cineole
6.40
37.6
7
p-cymene
7.07
2.5
8
α-pinene oxide
8.16
0.5
9
(±)-camphor
9.50
20.2
10
linalool
9.59
0.7
11
L-bornyl acetate
10.02
2.1
12
pinocarveol
10.58
0.3
13
iso-borneol
10.68
0.4
14
α-terpineol
10.88
1.7
15
(-)-borneol
10.94
1.7
total
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97.9
Table 2. In vivo insecticidal activities of rosemary essential oil and individual constituents to 3rd instar Trichoplusia ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells.
constituenta
LD50 (μg larva-1)
95% CLb
IC50
95% CL
(μg mL-1)
rosemary oil
215.8
191.2 - 246.1
54.6
1,8-cineole
229.6
171.3 - 341.6
>1000
(±)-camphor
471.3
402.4 - 538.2
>100
(+)-α-pinene
501.1
433.6 - 598.9
142.2
camphene
>1000
(-)-β-pinene
537.5
475.9 - 601.5
>1000
p-cymene
242.5
204.1 - 290.6
931.7
L-bornyl acetate
248.1
211.7 - 284.1
>300
(R)-(+)-limonene
233.8
158.5 - 367.6
49.8
92.4 - 104.2
61.9
51.2 - 74.6
498.3
371.2 - 669.1
α-terpineol
47.4 - 62.9
128.6 - 157.4
>100
(-)-borneol
713.1 - 1217.0
628.0 519.4 - 764.6 >100 There were ten constituents more than 1% of their concentrations in the test oil. b CL denotes confidence limit. a
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Table 3. Comparative insecticidal activity by topical application of four major constituents of rosemary essential oil in 3rd instar larvae of Trichoplusia ni ratio (%) 1,8-cineole (±)-camphor (+)-α-pinene camphene 63.4 36.6 72.0 28.0 83.9 16.1 59.8 40.2 75.0 25.0 66.9 33.1 50.8 29.4 19.8 56.5 32.7 10.8 63.2 24.6 12.2 49.9 33.5 16.6 46.3 26.8 18.0 8.9 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 25.0 25.0 25.0 25.0 a -1 observed LD50, μg insect .
obs LD50a 155.6 201.0 222.7 242.2 311.8 337.6 187.9 191.6 273.0 138.3 216.4 214.9 231.2 317.2 233.3 280.3 354.3 278.2 244.1 307.9 285.4 270.5
H&Pc 308.0 294.4 340.6 483.2 603.4 665.7 346.5 384. 5 380.6 569.0 405.6 342.6 357.6 607.0 486.1 735.6 750.5 391.5 556.1 566.0 650.8 546.6
b
expected LD50 based on each calculation model, μg insect-1.
c
Hewlett and Plackett’s calculation of expected LD50, μg insect-1.
d
Wadley’s calculation of expected LD50, μg insect-1.
e
Synergy ratio from Wadley’s calculation.
f
exp LD50b Wadleyd 267.7 255.0 245.1 482.7 542.9 600.2 294.8 290.5 280.5 528.1 314.4 294.4 300.0 352.7 485.7 640.5 667.6 344.8 388.8 395.3 592.1 408.6
Re 1.72 1.27 1.10 1.99 1.74 1.78 1.57 1.52 1.03 3.82 1.45 1.37 1.30 1.11 2.08 2.28 1.88 1.24 1.59 1.28 2.07 1.51
Sf S A A S S S S S A S A A A A S S S A S A S S
Determination of interaction of the mixture based on Wadley’s determination method, when R > 1.5 : synergistic, 1.5 ≥ R > 0.5 : additive, R ≤ 0.5 : antagonistic interaction.
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Figure 1. Standardd curve of florescence f intensity inn viable Triichoplusia ni ovarian cells. In the rangge of 5.6 × 104 to 7.4 × 105 cellss mL-1, it shhowed goodd correspon ndence betw ween the numberr of cells and florescencce intensity (R2 = 0.9716).
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Figure 2. 2 Mortality y and cytotooxicity caussed by artifi ficial rosemaary essentiaal oils blendded with full majjor constituents and onne compounnd missing on o 3rd instaar larvae of Trichoplusiia ni and the ovaarian cell linne of T. ni. A A: topical application a assay with rosemary oil’s o LD95 annd LD50 -1 of 446.77 and 215.88 μg insect , respectiv vely, B: fum migant assay y with rosem mary oil’s LC L 95 and -1 LC50 off 175.8 andd 107.4 μg mL air, reespectively, C: cytotox xicity assayy at 109.2 μg μ mL-1, which iis twice the IC50 of rossemary oil. Error E bars represent r standard erro or of the meean from three reeplications, and bars with w the sam me letter inddicate no siignificant differences (Tukey’s ( HSD teest, p < 0.055); upper annd lower caase letters refer r to the higher (LD D/LC)95) annd lower (LD/LC C50) concenttration levells in topicall applicationn and fumiggant assays.
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CONCLUSION: Based on our results, the insecticidal activity of rosemary oil appears to be a consequence of the synergistic interaction between 1,8-cineole and (±)-camphor, and (±)camphor should be considered a promising synergizing agent.
KEYWORDS: 1,8-cineole; (±)-camphor; synergy; toxicity; botanical insecticide
1 INTRODUCTION General concern about the risks associated with the repeated use of synthetic insecticides has led to searching for more environmentally benign and sustainable tools for pest control, as evidenced by the growth of organic produce in supermarkets.1 Botanical insecticides have long been touted as attractive alternatives to conventional pesticides because many botanicals pose little threat to the environment or to human health.2 The bioactivity of rosemary (Rosmarinus officinalis L.) essential oil has been reported for many insect and acarine pest species,3-7 and microorganisms including several food-borne pathogenic species of bacteria and fungi.8-9 Plant essential oils are complex mixtures of low molecular weight terpenoid compounds, and their chemical compositions can vary based not only on biological factors including genotype and interactions with other organisms, but also environmental factors such as climate, altitude, and nutrition levels. Chemical variation in rosemary essential oils has been well documented,10-12 and since these variations can influence biological activities, especially insecticidal activity,13 understanding the contribution of individual constituents of an essential oil as well as their interactions with respect to insecticidal activity is important for ‘quality control’ of a commercialized biopesticide based on this essential oil. In the present study, our This article is protected by copyright. All rights reserved
objective was to identify the contributions of individual essential oil constituents via different application methods, and assess any synergistic or antagonistic interactions in terms of insecticidal activity.
2 MATERIALS AND METHODS 2.1 Chemicals Rosemary essential oil (Intarome Fragrance & Flavors Corp. Norwood, NJ, USA) was obtained from EcoSafe Natural Product Inc. (Saanichton, BC, Canada). Standard compounds of constituents of the oil were purchased from Sigma-Aldrich (camphene [95%], (±)-camphor [96%], 1,8-cineole [99%], (R)-(+)-limonene [97%], (+)-α-pinene [98%], and (-)-β-pinene [99%], St Louis, MO, USA), and Thermo Fisher Scientific ((-)-borneol [97%], L-bornyl acetate [97%], p-cymene [≥99%] and α-terpineol [98%], Waltham, MA, USA). AlamarBlue® dye, Express Five® SFM medium, gentamicin and L-glutamine were purchased from Life Technologies (Carlsbad, CA, USA).
2.2 Gas chromatography-mass spectrophotometry Major constituents of rosemary oil were analyzed by the static headspace method on an Agilent 7890/5975C gas chromatograph inert XL mass spectrometer (Agilent Technologies Canada Inc., Ottawa, ON, Canada) operating in electron ionization mode fitted with a J&W DB-WAX 30 m × 0.25mm ID, 0.25 μm thickness fused silica column. The injection was done in a split mode (50:1) with the volume of 250 μL. The oven temperature setup was programed as 40 ºC for 1 min, followed by an increase of 10 ºC min-1 to 85 ºC, and then 35
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ºC min-1 to 250 ºC and held for 4 min with the total run time of 14.2 min. Helium was used as a carrier with 0.9 mL min-1 of flow. The data were analyzed using Enhanced Chemstation software and the constituents were identified by matching the spectra against the Wiley09/Nist08 MS library. The analysis was confirmed by re-analyzing an artificial essential oil, prepared by mixing the authentic standard compounds (> 1% in concentration) in their proportions found in the natural oil, under the same conditions. 2.3 Insect and cell line maintenance 2.3.1 Insect maintenance Eggs of the cabbage looper, Trichoplusia ni were obtained from the Great Lakes Forestry Centre, Natural Resources Canada (Sault Ste. Marie, ON, Canada). The colony was reared on pinto bean based artificial diet (Bio-Serv Inc., Frenchtown, NJ, USA) in the insectary at the University of British Columbia, Vancouver, BC, at 22-25 ºC and a 16:8 h LD photoperiod. 2.3.2 Cell line maintenance An ovarian cell line of T. ni (High Fivetm, Life Technologies) was grown in Express Five® SFM medium, supplemented with 1 mM L-glutamine and 50 μg mL-1 gentamicin. For long term maintenance of the cell line, subculture was performed twice a week with a split ratio of 1:11 to 2:10 (cell suspension : new medium) and cells were allowed to grow at 27 ºC in an incubation chamber.
2.4 Insecticidal and cytotoxic activity of essential oil and individual constituents 2.4.1 In vivo insecticidal activity A topical application method using third-instar larvae of the cabbage looper was conducted This article is protected by copyright. All rights reserved
using a Hamilton Microliter syringe attached to a repeating dispenser. Each larva received 1 μL of acetonic solution of the test compounds or acetone alone as the control. Three replications of ten treated larvae were each transferred into a 7 cm dia Petri dish with 0.5 g of artificial diet, and kept under the same conditions as for insect maintenance. After 24 h, mortality was recorded; larvae were determined to be dead when no movement was observed by probing the insect with a fine brush. Four to six different concentration levels were used to estimate LD50 values. 2.4.2 In vitro cytotoxicity T. ni cells were collected 72 h after the subculture and diluted to a density of 2 × 105 cells mL-1 with fresh medium. Fifty μL of cell suspension was loaded into a 96-well plate (Thermo Fisher Scientific), and allowed to settle for 2 h in the incubation chamber. Target compounds were dissolved in sterilized dimethyl sulfoxide (DMSO) and filtered with a nylon syringe filter with a pore size of 0.2 μm (Thermo Fisher Scientific), and then the filtered solution was mixed with fresh medium (1:149, v/v). Fifty μL of treatment solution was applied to each well of the plate, with DMSO/medium solution alone applied to control cells. After 48 h of incubation at 27 ºC, cells were inspected under Axiovert 35 inverted florescence microscope (Zeiss, Oberkochen, Germany) and viability of cells was monitored by a fluorescent method. Ten μL of alamarBlue® dye was added into each well, and fluorescence intensity was measured after 15 min of incubation using a Polarstar galaxy spectrophotometer (544 nm excitation and 590 nm emission, BMG Labtechnologies, Ortenberg, Germany). Nine concentrations with three replications were used to determine the IC50 values of essential oil and constituents. The intensity of the fluorescence was directly proportional to the number of metabolically active cells, and inversely proportional to the toxicity of the tested samples (Figure 1). This article is protected by copyright. All rights reserved
2.5 Comparative activities via different application methods To evaluate the individual constituents’ contribution to overall activity, a compound elimination assay was conducted using a method similar to that previously described. 7 A series of artificial essential oils was prepared with the constituents in their natural proportions, either as the full mixture or with one constituent missing. Each artificial oil had the same amounts of individual compounds as in the full mixture oil, with the amount of missing compound replaced by acetone. Two dose or concentration levels (LD/LC95 and LD/LC50) of each artificial oil were applied topically or by a fumigation method, and double the IC50 value was applied in the cytotoxicity assay. To determine the LC95 and LC50 values of rosemary essential oil by fumigation, ten third instar larvae were transferred into a 5.5 cm dia Petri dish with 0.5 g of artificial diet on a filter paper (4.25 cm diameter, Thermo Fisher Scientific) and covered with a ventilated lid (70 holes created by a heated syringe), and the edge was sealed with Parafilm (insect chamber). It was assumed that the larvae were exposed to the entire volatilized constituents of rosemary oil since the volume of the chamber was relatively small (25.3 mL). Six different concentrations of 50 μL of an acetonic solution of the essential oil were applied onto another filter paper. After 30 sec, the filter paper was attached to the bottom of a new Petri dish using adhesive tape (fumigant chamber), and both chambers were combined and sealed with Parafilm. Since the diameter of holes was smaller than that of the larvae, and there was a gap between the treated filter paper and the inner lid, larvae were prevented from having direct contact with the treated filter paper, but were exposed to the evaporated compounds. Mortality was determined after 24 h, and the compound elimination assay via fumigation was conducted in the same fashion. All tests were repeated three times. This article is protected by copyright. All rights reserved
2.6 Synergistic interactions among four major constituents To evaluate potential synergies between the four major constituents (1,8-cineole, (±)-camphor, (+)-α-pinene and camphene), mixtures were prepared following either the actual constituent ratio based on chemical analysis of the oil, or a 1:1 ratio of the compounds. Mixtures were applied topically to 3rd instar larvae, and their LD50 values were estimated after 24 h. To determine the relationships of the mixtures, we used two statistical models to compare expected and observed LD50 values, Hewlett and Plackett’s model14 and Wadley’s model.15 Based on Hewlett and Plackett’s calculation, the expected LD50 values (assuming additive interaction) were determined from the equation =
×
( )
+
×
( )
+
×
( )
+ ⋯ +
×
( )
where a was the proportion of compound A in the mixture, and LD50(a) was the LD50 of compound A. According to Wadley, theoretical LD50 values were calculated from + + + ⋅⋅⋅ +
= ( )
+
( )
+
( )
+ ⋅⋅⋅ +
( )
The interaction between the observed and theoretical LD50 values were compared as
=
expected LD observed LD
The relationship between the constituents of the mixture was defined as either synergistic (when R > 1.5), additive (1.5 ≥ R > 0.5) or antagonistic (R ≤ 0.5) based on this model.
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2.7 Data analysis Mortality and cytotoxicity data from the compound elimination assay were subjected to analysis of variance (ANOVA), and probit analysis was used to calculate the LD/LC95 and LD/LC50 values using StatPlus 2009 (version 5.8.4, AnalystSoft, Alexandria, VA, USA). IC50 values were determined by using GraphPad Prism 5 (Version 5.02, GraphPad Software Inc., La Jolla, CA, USA).
3 RESULTS 3.1 Chemical composition of rosemary essential oil Based on the GC-MS analysis of rosemary essential oil constituents, 1,8-cineole (37.6%) was the most abundant compound followed by (±)-camphor, (+)-α-pinene, and camphene (Table 1). These four major constituents comprised more than 80% of the total weight of the oil and all of the remaining individual constituents comprised less than 5% of the concentration, with a total of 97.7% of the constituents identified.
3.2 Insecticidal activity and cytotoxicity of individual constituent In third instar larvae of the cabbage looper, the LD50 value of rosemary oil was 215.8 μg insect-1 (Table 2). Among the constituents tested, α-terpineol was the most potent (61.9 μg insect-1), whereas camphene was inactive at the highest concentration treated (> 1,000 μg insect-1). Although 1,8-cineole showed activity comparable to the complete oil, none of the major compounds (> 5% of the oil) had LD50 values lower than that of the intact oil. This article is protected by copyright. All rights reserved
In the in vitro cytotoxicity assay, the most active compound was (R)-(+)-limonene (49.8 μg mL-1), and several constituents including 1,8-cineole (> 1,000) and (±)-camphor (> 100) failed to show a cytotoxic effect on even at the highest concentrations tested.
3.3 Contribution of individual compound to overall activity At the higher concentration of topical application (LD95 of rosemary oil), there was a significant decrease in mortality when 1,8-cineole was eliminated from the artificial oil (Figure 2A). Although mortality decreased slightly when (±)-camphor was excluded, it was not statistically different (P < 0.05). In the fumigation assay against 3rd instar larvae of T. ni, rosemary essential oil gave 175.8 and 107.4 μg mL-1 air as LC95 and LC50 values, respectively. Based on these concentrations, the compound elimination assay revealed that 1,8-cineole and (±)-camphor were significantly more active than all the other constituents (P < 0.05, Figure 2B). In the cytotoxicity assay, the most significant decrease of activity occurred when (+)-αpinene was eliminated, but the two major constituents, 1,8-cineole and (±)-camphor did not decrease the cytotoxic effect when they were missing (Figure 2C).
3.4 Interactions among mixtures Insecticidal synergistic interactions among the four major constituents (1,8-cineole, (±)camphor, (+)-α-pinene and camphene) were investigated (Table 3). Combinations following natural proportions of the essential oil composition showed several synergistic relationships; the most significant synergy based on Wadley’s determination was the mixture of (±)This article is protected by copyright. All rights reserved
camphor+(+)-α-pinene+camphene (R = 3.82). A binary mixture of the two most abundant constituents, 1,8-cineole+(±)-camphor, produced a significantly lower LD50 than for the individual compounds. A notable trend of synergy was found with (±)-camphor. Apart from the full mixture of the four compounds, all other combinations that included (±)-camphor as an element showed synergy (R > 1.5). When the constituents were mixed at 1:1 ratio, (±)-camphor+camphene showed the greatest increase in toxicity (R = 2.28), followed by (±)-camphor+(+)-α-pinene and (±)-camphor+(+)α-pinene+camphene. No combination tested showed an antagonistic interaction.
4 DISCUSSION Our compound elimination bioassays pointed to 1,8-cineole as the major insecticidal constituent of rosemary oil based on topical administration, and 1,8-cineole and (±)-camphor as major insecticidal constituents via fumigation to the cabbage looper. Although α-terpineol had the lowest LD50 value when applied topically, its concentration in the oil is relatively small (1.7%), and elimination of it from the artificial mixture did not lead to a decrease in mortality, suggesting that its contribution to overall toxicity is limited. In a previous study of our laboratory of the toxicity of rosemary oil to the two-spotted spider mite, Tetranychus urticae,7 1,8-cineole showed the greatest contribution to overall activity, followed by (+)-αpinene. In another study of basil essential oil (Ocimum kilimandscharicum), the major constituent, (±)-camphor, exhibited not only high insecticidal activity but was also significant repellent and inhibited growth and development of four species of stored product beetles.16 It is often observed that crude essential oils are considerably more active than any of their individual constituents.17-19 Our results clearly indicated several synergistic interactions This article is protected by copyright. All rights reserved
between constituents of rosemary oil; all combinations of the major constituents tested produced toxicity comparable to or greater than that expected. The two major constituents, 1,8-cineole and (±)-camphor are synergistic (LD50 = 155.6 μg insect-1, R = 1.72). Taking into consideration concentrations of these constituents in the essential oil (57.8% combined) and their enhanced toxicity when combined, the insecticidal activity of rosemary oil might be mainly a consequence of their synergistic interaction. Since plant essential oils obtained from ‘actual’ plants vary chemically based on numerous biotic and abiotic factors, the chemical profile of oils will not be uniform, except where oils from different sources are intentionally blended to achieve a consistent composition. We previously reported variation in response of noctuid larvae based on variation in chemical composition of rosemary oil,13 with LD50 values ranging from 58.9 to 335.9 μg insect-1 in T. ni, and 167.1 to 372.1 μg insect-1 in the armyworm, Pseudaletia unipuncta. This points to the importance of understanding the contributions of individual constituents and combinations thereof to overall insecticidal activity as a basis for standardization and quality control of commercial insecticides based on plant essential oils. Interestingly, among the four major constituents of rosemary oil, (±)-camphor displayed the most consistent synergistic effects with other compounds. Eleven out of 14 combinations that included (±)-camphor showed synergy, and the remaining 3 combinations showed at least 20% increases in insecticidal activities (Table 3). In a similar study of synergy among 30 insecticidal compounds to Spodoptera littoralis, (±)-camphor exhibited synergy with 22 substances, and 9 of the mixtures with (±)-camphor showed the maximal synergic effect.20 It is still unclear how this synergy is achieved, but based on pharmacological studies Wagner and Ulrich-Merzenich suggested four possible mechanisms; (a) synergistic multi-target effects, (b) pharmacokinetic or physicochemical effects based on improved solubility and
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absorption rate, (c) interactions of agents with resistance mechanisms, and (d) respective elimination or neutralization of adverse effects.21 Since plant essential oils are usually complex mixture of compounds, they can be expected to have multiple modes of action. For example, 1,8-cineole has been reported to have inhibitory activity on AChE, associated with octopamine
23
and GABA receptors.
24
22
but it is also
Also, some essential oils have been
shown to inhibit detoxifying enzymes, resulting in synergy with synthetic insecticides.
25
To
date, increased cuticular penetration by essential oil constituents as a mechanism of synergy has not been reported, but increased penetration by enhanced solubility might also be involved as the constituents often have a wide range of hydrophilic-hydrophobic properties. Synergistic insecticidal activities have been observed in many cases, not only between constituents of essential oils, but also between essential oils, synthetic insecticides,
28
26
monoterpene compounds,
and between essential oils and synthetic compounds.
29
27
Several
statistical models have been proposed to determine synergy, and in the present study we adopted two models, Hewlett and Plackett’s calculation and Wadley’s. Some of the estimated LD50 values were similar, but in general, Wadley’s model was deemed to be the more conservative of the two. One shortcoming of these two models is the time and effort required to obtain the empirical LD50 values of the individual compounds as well as those of mixtures. A somewhat more convenient model is Finney’s calculation.
30
Expected mortality of the
mixture can be calculated using the equation: E = Oa + Ob (1 – Oa), where E is the expected mortality, Oa and Ob are the observed mortalities of the individual compounds. A chi-square value is to be obtained via: χ2 = {(Om – E)2} / E, where Om is observed mortality of the mixture. The calculated chi-square can be compared to a chi-square tabular value with df = 1 and α = 0.05, which gives 3.84. If the chi-square is > 3.84, synergy is confirmed. However, caution must be used with this approach. Being based on single concentrations or doses, this
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method is considerably less precise than models based on LD50 values, and depends on appropriate selection of the single doses used. We found that neither 1,8-cineole or (±)-camphor nor their mixture had a potent cytotoxic effect on the T. ni cell line (IC50 > 1,000 μg mL-1, > 100 μg mL-1, > 1,000 μg mL-1, respectively). One of the possible reasons for this could be the lower solubility of the compounds in the cytotoxicity assay system, because we used a relatively low concentration of DMSO as a carrier, and no surfactant. While it is possible that some of the test compounds were not fully in solution at the higher concentrations tested, we believe that at lesser concentrations the compounds would have been completely solubilized. Insect cell systems have been investigated as a convenient tool to screen for insecticidal bioactivity, as a replacement for bioassays with live insects. However, the rapid insecticidal action of plant essential oils to some insect species suggests one or more neurotoxic modes of action. 2 It has been reported that certain biochemical targets such as octopamine 31 or GABA 32
receptors are associated with the insecticidal mode of action of monoterpene compounds.
Decombel et al. 33 reported weak correspondence between in vivo insecticidal activity and in vitro cytotoxicity of synthetic neurotoxic insecticides. Moreover, no direct association (high mortality to mites / high cytotoxicity to an insect cell line) was found in a screening study of 67 plant extracts. 34 Cell culture systems may be useful in identifying prospective insecticides with modes-of-action based on disruption of primary metabolism, protein synthesis and other activities common to most animal cells, but not for substances that depend on receptors found only in the nervous system (i.e., those with a neurotoxic mode of action). It has been more than 15 years since essential oil-based pesticides were introduced into the US market. EcoSMART Technologies is the leading company specialized in this area, having introduced a range of agricultural, professional and consumer botanical insecticides using This article is protected by copyright. All rights reserved
rosemary oil and other essential oils derived from clove leaf (Syzygium aromaticum), peppermint (Mentha × piperita), cinnamon leaf (Cinnamomum zeylandicum), lemongrass (Cymbopogon nardus) and thyme (Thymus vulgaris) as active ingredients.
35
Understanding
synergistic mechanisms within and between essential oils and their constituents may facilitate their further commercial development toward more favorable pest management products.
ACKNOWLEDGMENTS The authors gratefully acknowledge Nancy Brad, Zyta Abramowski and Lina Madilao for technical supports. This work was supported by a University of British Columbia graduate fellowship [to JHT] and a Discovery grant from the Natural Sciences and Engineering Research Council of Canada [2729-11] [to MBI].
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Sitophilus oryzae (L.) (Coleoptera : Curculionidae). Research Journal of Biological Sciences 2:75-80 (2007). 27 Pavela R, Acute and synergistic effects of monoterpenoid essential oil compounds on the larvae of Spodoptera littoralis. J Biopestic 3:573-578 (2010). 28 Pennetier C, Corbel V and Hougard JM, Combination of a non-pyrethroid insecticide and a repellent: a new approach for controlling knockdown-resistant mosquitoes. Am J Trop Med Hyg 72:739-744 (2005). 29 Shaalan EAS, Canyon D, Younes MWF, Abdel-Wahab H and Mansour AH, A review of botanical phytochemicals with mosquitocidal potential. Environ Int 31:1149-1166 (2005). 30 Trisyono A and Whalon ME, Toxicity of neem applied alone and in combinations with Bacillus thuringiensis to Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 92:1281-1288 (1999). 31 Enan EE. Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to essential oils. Arch Insect Biochem Physiol 59:161-171 (2005). 32 Tong F and Coats JR, Effects of monoterpenoid insecticides on [3H]-TBOB binding in house fly GABA receptor and 36Cl− uptake in American cockroach ventral nerve cord. Pestic Biochem Physiol 98:317-324 (2010). 33 Decombel L, Smagghe G and Tirry L, Action of major insecticide groups on insect cell lines of the beet armyworm, Spodoptera exigua, compared with larvicidal toxicity. In Vitro Cell Dev Biol-Animal 40:43-51 (2004).
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Table 1. Chemical constituents of rosemary essential oil no.
constituent
retention time (min)
% area
1
(+)-α-pinene
4.19
15.7
2
α-fenchene
4.62
0.2
3
camphene
4.72
7.9
4
(-)-β-pinene
5.22
4.3
5
(R)-(+)-limonene
6.29
2.1
6
1,8-cineole
6.40
37.6
7
p-cymene
7.07
2.5
8
α-pinene oxide
8.16
0.5
9
(±)-camphor
9.50
20.2
10
linalool
9.59
0.7
11
L-bornyl acetate
10.02
2.1
12
pinocarveol
10.58
0.3
13
iso-borneol
10.68
0.4
14
α-terpineol
10.88
1.7
15
(-)-borneol
10.94
1.7
total
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97.9
Table 2. In vivo insecticidal activities of rosemary essential oil and individual constituents to 3rd instar Trichoplusia ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells.
constituenta
LD50 (μg larva-1)
95% CLb
IC50
95% CL
(μg mL-1)
rosemary oil
215.8
191.2 - 246.1
54.6
1,8-cineole
229.6
171.3 - 341.6
>1000
(±)-camphor
471.3
402.4 - 538.2
>100
(+)-α-pinene
501.1
433.6 - 598.9
142.2
camphene
>1000
(-)-β-pinene
537.5
475.9 - 601.5
>1000
p-cymene
242.5
204.1 - 290.6
931.7
L-bornyl acetate
248.1
211.7 - 284.1
>300
(R)-(+)-limonene
233.8
158.5 - 367.6
49.8
92.4 - 104.2
61.9
51.2 - 74.6
498.3
371.2 - 669.1
α-terpineol
47.4 - 62.9
128.6 - 157.4
>100
(-)-borneol
713.1 - 1217.0
628.0 519.4 - 764.6 >100 There were ten constituents more than 1% of their concentrations in the test oil. b CL denotes confidence limit. a
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Table 3. Comparative insecticidal activity by topical application of four major constituents of rosemary essential oil in 3rd instar larvae of Trichoplusia ni ratio (%) 1,8-cineole (±)-camphor (+)-α-pinene camphene 63.4 36.6 72.0 28.0 83.9 16.1 59.8 40.2 75.0 25.0 66.9 33.1 50.8 29.4 19.8 56.5 32.7 10.8 63.2 24.6 12.2 49.9 33.5 16.6 46.3 26.8 18.0 8.9 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 33.3 25.0 25.0 25.0 25.0 a -1 observed LD50, μg insect .
obs LD50a 155.6 201.0 222.7 242.2 311.8 337.6 187.9 191.6 273.0 138.3 216.4 214.9 231.2 317.2 233.3 280.3 354.3 278.2 244.1 307.9 285.4 270.5
H&Pc 308.0 294.4 340.6 483.2 603.4 665.7 346.5 384. 5 380.6 569.0 405.6 342.6 357.6 607.0 486.1 735.6 750.5 391.5 556.1 566.0 650.8 546.6
b
expected LD50 based on each calculation model, μg insect-1.
c
Hewlett and Plackett’s calculation of expected LD50, μg insect-1.
d
Wadley’s calculation of expected LD50, μg insect-1.
e
Synergy ratio from Wadley’s calculation.
f
exp LD50b Wadleyd 267.7 255.0 245.1 482.7 542.9 600.2 294.8 290.5 280.5 528.1 314.4 294.4 300.0 352.7 485.7 640.5 667.6 344.8 388.8 395.3 592.1 408.6
Re 1.72 1.27 1.10 1.99 1.74 1.78 1.57 1.52 1.03 3.82 1.45 1.37 1.30 1.11 2.08 2.28 1.88 1.24 1.59 1.28 2.07 1.51
Sf S A A S S S S S A S A A A A S S S A S A S S
Determination of interaction of the mixture based on Wadley’s determination method, when R > 1.5 : synergistic, 1.5 ≥ R > 0.5 : additive, R ≤ 0.5 : antagonistic interaction.
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Figure 1. Standardd curve of florescence f intensity inn viable Triichoplusia ni ovarian cells. In the rangge of 5.6 × 104 to 7.4 × 105 cellss mL-1, it shhowed goodd correspon ndence betw ween the numberr of cells and florescencce intensity (R2 = 0.9716).
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Figure 2. 2 Mortality y and cytotooxicity caussed by artifi ficial rosemaary essentiaal oils blendded with full majjor constituents and onne compounnd missing on o 3rd instaar larvae of Trichoplusiia ni and the ovaarian cell linne of T. ni. A A: topical application a assay with rosemary oil’s o LD95 annd LD50 -1 of 446.77 and 215.88 μg insect , respectiv vely, B: fum migant assay y with rosem mary oil’s LC L 95 and -1 LC50 off 175.8 andd 107.4 μg mL air, reespectively, C: cytotox xicity assayy at 109.2 μg μ mL-1, which iis twice the IC50 of rossemary oil. Error E bars represent r standard erro or of the meean from three reeplications, and bars with w the sam me letter inddicate no siignificant differences (Tukey’s ( HSD teest, p < 0.055); upper annd lower caase letters refer r to the higher (LD D/LC)95) annd lower (LD/LC C50) concenttration levells in topicall applicationn and fumiggant assays.
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