J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 387 http://jad.tums.ac.ir Published Online: December 25, 2018 Original Article Chemical Composition and Repellency of Origanum vulgare Essential Oil against Cimex lectularius under Laboratory Conditions Mona Sharififard 1, 2, Ismaeil Alizadeh 1, 2, *Elham Jahanifard 1, 2, Changlu Wang 3, Mohammad Ebrahim Azemi 4 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 3Department of Entomology, Rutgers University, New Brunswick, NJ, USA 4Department of Pharmacognosy, Medicinal Plant and Natural Product, Research Center, School of Pharmacy, Jundishapur University of Medical Sciences, Ahvaz, Iran (Received 16 Sep 2017; accepted 27 June 2018) Abstract Background: The common bed bug (Cimex lectularius), a nocturnal blood-feeding ectoparasite, is considered an urban pest with public health importance in human environments. We aimed to determine the repellency effect of oregano essential oil, Origanum vulgare, against this pest under laboratory conditions. Methods: The essential oil was prepared from dried leaves using hydro-distillation method. A gas chromatography- mass spectrometer (GC-MS) was used for analysis and identification of oregano essential oil compounds. Treated surface method in Petri dish was carried out to evaluate repellency potential of the oil using 4th and 5th instar nymphs and adults. The concentration-repellency response of oregano essential oil was calculated and compared with a commercial insect repellent stick containing 33% N, N-diethyl-meta-toluamide (DEET). Results: The oregano essential oil consisted of 158 compounds with terpineol (22.85%) and α–terpinene (20.60%) being the major components by volume. The EC50 and EC99 of oregano oil (effective concentrations causing 50% and 90% repellency of bed bugs) were 1.61 and 6.57mg/cm2 at 9h after application, respectively. The 40% oregano es- sential oil exhibited 100% repellency against bed bug at 3, 5, 9 and 24h after application while the repellency index of DEET 33% was 100% at 3 and 5h and it decreased to 80% and 27% at 9 and 24 hours. Conclusion: Oregano oil 40% exhibited more repellency compared to commercial insect repellent stick containing 33% DEET. Further studies are warranted to confirm the effectiveness of oregano essential oil in personal protecting against bed bug biting. Keywords: Cimex lectularius, Essential oil, Origanum vulgare, repellency Introduction Bed bug (Cimex lectularius Linnaeus), is a nocturnal blood-feeding ectoparasite consid- ered as urban, common and nuisance pest in the recent past (1, 2). This insect affects every- one in different social status and infests eve- rywhere. The case of infestation has been re- ported from countries including United King- dom, Denmark, Norway, Italy, Spain, Sweden, Scandinavia, Switzerland, Australia, Brazil, Iran, Thailand, Malaysia, Singapore, Kuwait, Nigeria and the United States (2–11). The bed bug infestation became rare with the new pest control techniques in North America and Eu- rope a century ago (12), but the United States has significant resurgence as a perfect storm in 2006 (13). The global resurgence of bed bug infestations was related to increased levels of international transport through tourism and *Corresponding author: Dr Elham Jahanifard, E-mail: jahanifard-e@ajums.ac.ir, elham.jahani56@gmail.com mailto:jahanifard-e@ajums.ac.ir J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 388 http://jad.tums.ac.ir Published Online: December 25, 2018 trade, immigration insecticide resistance and possibly increased temperatures (14). Besides pain and itchiness, the problems caused by bed bug infestations include psychological distress with nightmares, insomnia, anxiety, social iso- lation, and quality of life (15). Bed bugs hide in protected places such as crevices on beds and other furniture, inside devices, inside walls, floor cracks. This par- tially explains why they are difficult to detect and control (16). Surface treatment with pyre- throids and other classes of insecticides, phys- ical control, and personal protection are the main recommendations for the control and prevention of bed bugs infestation (1, 17, 18). Application of insecticides poses an imme- diate risk to human health and the environ- ment especially when insecticides are applied on furniture (17). Personal protection using repellent com- pounds on human skin is an effective and in some cases the only practical approach to the control of biting insects. The most common synthetic repellent used on blood-sucking arthropods is N, N-diethyl-meta-toluamide (DEET) produced in 1954 (19, 20). Due to reports of allergic and toxic effects especial- ly on children and pregnant women from DEET, it is placed under investigation (21– 23). Therefore, finding a safer repellent com- pound for use on human body surface is nec- essary. Botanical essential oils are regarded as environmentally friendly products, ecolog- ical alternatives and low mammalian toxicity materials (24). However, some plant-based repellents should be used with caution due to their compounds (25). Essential oils have been introduced as green pesticides and most of them are non-toxic to humans, animals and are safe and friendly to the environment (26). These products are good alternatives to synthetic insecticides and can delay the development of resistance to insect pests (27). Essential oils (EO) are secondary metabolites of plants. They are complex mix- tures of volatile organic compounds such as hydrocarbons and oxygenates (28, 29). To- day, many essential oil products have been developed and are effective as repellents or surface spraying compounds on pest of med- ical important (17, 30). Essential oils have var- ious activities against insect pests, including insecticidal, antifeedant, repellent, oviposition, deterrent and growth regulatory (30, 31). Re- pellent activity of essential oils is an effec- tive way of controlling biting insects includ- ing bed bugs and they can be applied on lug- gage, fabric materials and furniture in order to reduce bed bug infestation by preventing bed bugs into their home (27). Oregano oil is considered as an excellent antiseptic and insect repellent. It has some active ingredients such as carvacrol, thymol and α-terpinene reported being highly effec- tive in repelling mosquitoes (32, 33). More- over, it showed significant repel activity at concentration range of 2.5–30% against Su- pella longipalpa (34). The aim of this study was to determine the repellency activity of oregano essential oil (O. vulgare) on bed bugs in comparison with DEET against bed bugs (C. lectularius). Materials and Methods Bed Bugs The adult and nymph stages of C. lectu- larius were collected from infested homes in Ahvaz City, southern Iran and transferred to the Laboratory of Medical Entomology De- partment, School of Public Health, Ahvaz, Jundishapur University of Medical Sciences, Ahvaz, Iran. The insects were reared in plas- tic containers (12cm in height and 6cm in di- ameter) with folded papers as harborage and kept at 26±1 °C, 50±5% relative humidity (RH), photoperiod of 12:12 (L: D) (17, 27). The colony of C. lectularius were fed weekly on rabbits and allowed to suck blood for 10min (2). The bed bugs were starved for 7 d before bioassays. J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 389 http://jad.tums.ac.ir Published Online: December 25, 2018 Chemical compounds Insect repellent stick (33% DEET) as com- mercial formulation was purchased from Rey- han Naghsh Jahan Pharmaceutical Cosmetic and Hygienic Company. It is used widely in Iran on blood-sucking insects such as mosqui- toes, sand flies, ticks and bed bugs. Essential oil isolation Oregano plant (O. vulgare) was collected from its natural habitat from Yazd Province, central part of Iran (latitudes 54.20257 and lon- gitudes 32. 00315). Collected Oregano spec- imens were identified by the Department of Pharmacognosy, Ahvaz Jundishapur Univer- sity of Medical Sciences. Firstly, fresh leaves dried with air, and next 200gr of oregano was mixed with 400ml of distilled water and placed in a 1L flask. In the following the essential oil oregano used in the assay was isolated from dried fresh leaves by the hydrodistillation meth- od using a Clevenger-type apparatus (Model BP, British Pharmacopoeia, Manufacturer Py- rex Fan Company, Iran and mantle model H610 manufacturer Fater Company, Iran) at 90±5 for 5 hours. We extracted 0.9cc pure (about 100%) essential oil of 100gr of dried leaves of O. vulgare. Sodium sulfate was used for de- hydration. The extracted essential oils were stored at 4 °C in dark glass vials for further ex- periment (35). Gas chromatographic-mass spectral analysis Gas-chromatography-mass spectrometer (GC-MS) was used for the analysis and iden- tification of oregano essential oil compounds (Hewlett-Packard 6890, Agilent Technology, Santa Clara, California, USA). It is equipped with HP–5MS column (30m× 0.25mm× 0.25 µm). The initial temperature used was 40 °C for 1min and was later raised to 220 °C at a rate of 3 °C/min and finally raised to 270 °C for 5min at a rate of 20 °C/min. Other param- eters of the GC-MC machine included carrier gas Helium (99/999%), injector temperature (260 °C), detector temperature (FID, 270 °C), split-less mode, ionization potential of 70eV, scan rate of 1 scan/sec, the scan range of m/z 40–48 was used for all analysis. The essential oil constituents were identified by comparing their retention indices, mass spectra fragmen- tation with those in a stored Wiley 7n.1 mass computer library and those of National In- stitute of Standards and Technology (NIST) (36). Petri dish repellency assay Oregano essential oil at concentrations of 0.625, 1.25, 2.5, 5, 10, 20 and 40% (V/V) were evaluated against the laboratory-reared bed bugs in plastic Petri dishes and compared with DEET (33%). Moreover, the concentrations were calculated as doses of 0.1, 0.21, 0.43, 0.86, 1.72, 3.45, 6.9mg/cm2 for drawing dose- response curve. Plastic Petri dishes of 8cm di- ameter by 1.5cm height were used in this exper- iment. Ethanol was used as solvent for es- sential oil. White filter papers were divided into two equal halves and inserted into the bot- tom part of the Petri dishes. A piece of fold- ed filter paper was placed in the middle as bed bug harborage (Fig. 1). This part (bed bug harborage) was treated with 0.7ml of essential oil solution using a micro-sampler and the oth- er half was left untreated (27). In the control (negative and positive) plastic Petri dishes group, one half of the filter paper and the harborage were treated with 0.7ml of 96% eth- anol or 33% DEET, and the other half was left untreated. Ten-bed bugs (4th and 5th in- stars, nymphs and adults) for each treatment group were released into the center of each Petri dish (Fig. 1). The number of bed bugs on each side of the Petri dish was recorded at 3, 5, 9, and 24h post treatment by visual in- spection. The experiments were replicated four times for any concentrations in oregano essential oil, Ethanol, and 33% DEET. All Petri dishes were kept at 26±2 °C, photoperiod of 12:12 (L: D) and 55±5% relative humidity (RH). J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 390 http://jad.tums.ac.ir Published Online: December 25, 2018 Statistical Analysis Probit analysis was used to calculate the effective concentration (EC50 and EC99). Re- pellency indices were calculated using: repel- lency =(C–T)/C×100, where C is the mean num- ber of bed bugs located on the treated filter paper in all control plastic Petri dishes, and T is the number of bed bugs located in half part of the treated filter paper at unfixed concentra- tion test in the plastic Petri dishes (27). Re- pellency indices were compared using analy- sis of (ANOVA) followed by Dunnett test to distinguish between the treatments. All statis- tical analyses were performed using SPSS ver. 16 (Chicago, IL, USA). Results Yields and chemical constituents of essen- tial oil The content of essential oil was obtained from 0.8 to 1ml per 100gr of dried leaves and the density of the essential oil was calculated as d EO= 0.94g mL -1. Oregano essential oil was found to contain 158 compounds using GC- MS. The most common compounds are shown in Table 1 and Fig. 2. In addition, chemical analysis of essential oil of O. vulgare is shown in Fig. 3. Dose-response of essential oil The results of dose-response test are showed with the calculation of repelling ef- fective concentration as mg essential oil per cm2 (EC50 and EC99) in Table 2. The dose-re- sponse data was calculated from repels ef- fective concentration of essential oil in mg per cm2 of the surface. The data obtained from 9h exposure time was important because this time represents the typical time period when peo- ple are in bed and exposure to common bed bug biting. They were 1.61 and 6.57mg/cm2 for EC50 and EC99 at 9h after exposure, respec- tively with slope (±SE) = 0.469±0.073 and Chi- square (df)= 17.17 and P< 0.904. Calculated dose-response curve for Oregano essential oil after 9h is shown in Fig. 4. Bed bug repellency activity of essential oil Repellency indices of oregano essential oil and 33% DEET on C. lectularius are shown in Table 3. The repellency means are notice- able even in very low concentrations at 3h after exposure. The Dunnett test showed, there was no significant difference in repellency be- tween 33% DEET and oregano essential oil in the concentrations of 10, 20 and 40% (P= 0.4). After 5h, the repellent activity of ore- gano oil was similar to 33% DEET at the concentrations of 10% to 40% and it varied between 86% and 100%. The repellency of oregano oil at the concentrations of 10% and 20% was still obvious (86–93% repellency). Significant difference in repellency was ob- served between 0.625%, 1.25%, and 2.5% con- centrations of oregano essential oil and 33% DEET after 3, 5 and 24h (P< 0.001), while the differences in repellency of 5%, 10%, 20%, and 40% oregano essential oil with 33% DEET were not significant after 3 and 5h (P= 0.25). The concentration of 40% oregano essential oil had 100% repellency against bed bugs at 3, 5 and 24h post exposure, while the repel- lency index of 33% DEET decreased to 27% after 24h (Table 3). The repellency of ore- gano essential oil at concentrations of 0.625– 20% significantly declined from 3h to 24h, but it was constant at a concentration of 40% and exhibited similar repellency to 33% DEET at a concentration of 20%. At 9h after exposure, significant differ- ences were observed between repellency of 0.625%, 1.25%, 2.5%, and 5% concentrations of Oregano essential oil and 33% DEET (P< 0.001), while the differences between 10%, 20%, and 40% oregano essential oil and 33% DEET were not significant (P=0.21) (Fig. 5). J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 391 http://jad.tums.ac.ir Published Online: December 25, 2018 Table 1. Constituents of oregano essential oil by GC-MS analyses Compound RI * Major Constituents )%( Pinene 939 1.15 γ-Terpinene 1056 0.14 α – Terpinene 1016 20.60 α – Terpinolene 1186 2.16 Terpineol 1192 22.85 Thymol 1290 4.53 Carvacrol 1299 4.9 Caryophyllene 1419 6.75 Other compounds - 36.92 *RI, retention index as determined on an HP-5MS column using the homologous series of n-hydrocarbons Table 2. Effective doses of Oregano essential oil against Cimex lectularius by treated surface method in Petri dishes Time EC50 (CL*) mg/cm2 EC99 (CL*) /cm2 Slope (±SE) Chi-Square ** (df) P value After 3h 0.27(0.05-0.42) 1.96 (1.52-2.97) 1.377 (±0.279) 21.57 (26) 0.712 After 5h 0.87(0.57-1.16) 4.10 (3.31-5.55) 0.720 (±0.112) 31.70 (26) 0.203 After 9h 1.61(1.18-2.09) 6.57 (5.29-8.92) 0.469 (±0.073) 17.17(26) 0.904 After 24h 4.53 (***) 6.10 (***) 1.19 (±0.877) 12.70 (26) 0.986 EC50 and EC99: Effective concentration cause 50% and 99% repellency against Bed Bug. *CL: confidence limits. **Since Chi-square goodness of fit test is not significant (P> 0.05), no heterogeneity factor is used in the calculation of confidence limits. ***Probit model did not work because <25% repellency occurred. Table 3. Repellency index of essential oil, ethanol and 33% DEET against bed bugs Treatment Concentration (%) Repellency index (Mean%±SE) 3h 5h 9h 24h 0.625 20±0.88a 14±1.15a 13±1a 3±0.33a 1.25 32±0.57ab 27±1.33ab 27±0.33a 7±0.57a Oregano essential oil 2.5 44±0.88bc 39±0.33bc 34±0.88ab 10±0.33a 5 68±0.33cd 61±0.33cd 42±0.57abc 14±0.33ab 10 100±0.0d 86±0.33d 65±0.57bcd 17±0.57b 20 100±0.0 d 93±0.66d 73±0.33c 25±0.57b 40 100±0.0 d 100±0.0d 100±0.0d 100±0.0c 33% DEET 33 100±0.0 d 100±0.0d 100±0.0d 27±0.57b Ethanol 96 - - - - Values in columns followed by different letters are significantly different (P< 0.05) J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 392 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 1. Petri dish repellency assay of oregano essential oil, ethanol and 33% DEET on bed bugs Fig. 2. Dose-response curve for Oregano essential oil after 9 hour in laboratory assay Fig. 3. Chemical structures of major compounds of the essential oil of Origanum vulgare J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 393 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 4. A typical GC-MS chromatogram showing the chemical analysis of essential oil from Origanum vulgare Fig. 5. Repellency of ethanol, different concentrations of Oregano essential oil and 33% DEET against bed bugs in Petri dish assays. Different letters above the bars indicate significant differences at α= 0.05 Discussion In this study, terpineol (22.85%) was the most abundant chemical component of orega- no essential oil followed by α-terpinene with 20.6% (v/v). The component of α-terpinene in O. vulgare collected from Mazandaran was 1.018% (38). The plant phenology and com- position was likely affected by ecologic con- ditions. The percentage of carvacrol obtained 4.9% in the present investigation. The percent- age of carvacrol, collected from various local- ities in West Azarbaijan Province of Iran were reported in the range of 23.54% to 67.09% (39). The chemical composition of oregano oil changed due to plants’ development, the popu- J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 394 http://jad.tums.ac.ir Published Online: December 25, 2018 lation or variation, and the cultivation condi- tions. The content of carvacrol depends on cli- matic condition and also it is can be variable in budding period and in full bloom (40). In addition, difference in the chemical composi- tion of oregano oil from previously published papers with our study may is regarding the type of oregano subspecies. Two percent of α-terpinene, monoterpenes derived from Thy- mus vulgaris were reported with stronger re- pellency activity than DEET against Culex pipiens (33). In our findings, oregano essen- tial oil showed more potential as repellent com- pound than 33% DEET, a commercial prod- uct, against bed bugs. The high concentration α-terpinene may be responsible for the oil’s repellency. The aim of using repellant compound to- wards hematophagous pest is to reduce the frequency of biting in endemic areas and pre- vent bites in areas where allergic reactions to biting insects are common (41). Very few stud- ies have been conducted to evaluate insect re- pellent activities of essential oils against bed bugs (17, 27). More tolerance of bed bugs against insect repellents were compared with other blood-sucking insects (27). According to the Petri dish repellency as- say, the oregano essential oil of 2.5% showed 10% repellency after 24h. While the same con- centration of oil demonstrated 99.1% repel- lent activity on the brown-banded cockroach (Supella longipalpa) after the same time (34). The sub-lethal concentrations (LC10 and LC30) of oregano essential oil had an average repel- lency of 22.8% and 49.8% against the diamond- back moth (Plutella xylustella) respectively (42). Insect species, method of test, formula- tion, and concentrations of the oil were re- sponsible for the different results observed in the reported investigations. However the re- pellency indices were different between se- lected concentrations of oregano oil and there was no significant difference between effica- cy of DEET (33%) with 20% oregano oil at 3, 5, 9h post exposure. No repel activity was observed in the control group of our study and all the bed bugs stayed under harborage at 3, 5, and 9h post ethanol exposure. Our investigation showed that repellency of 40% oregano oil was 100% against the com- mon bed bugs in Petri dish test. It repellency index was similar to 33% DEET at 3 and 5h and it was more effective than 33% DEET at 9h after application. The 40% concentration of oregano oil was found to be safer for ap- plication in human environments and also for personal protection. While EcoRaider (1% ge- raniol, 1% cedar extract, and 2% sodium lau- ryl sulfate) and bed bug patrol (0.003% clove oil, 1% peppermint oil, and 1.3% sodium lau- ryl sulfate) as botanical repellents did not ex- hibit detectable repellency against bed bug in the presence of carbon dioxide source (17). DEET, picaridin, tea tree oil, peppermint oil, and citronella oil were tested for repellency activity on Triathoma rubida using small re- strained rats. No long-range repellency was ob- served. Only citronella oil was able to stop all probing and feeding of T. rubida and appears to be a promising potential repellent to prevent sleeping people from being bitten by kissing bugs (41). The efficacy of any pesticide or re- pellent compound can vary with the testing method, rate of application, bed bug strain and physiological stage (17). Conclusion Preferably, the EC99 dose should be esti- mated to determine the protection time against blood-sucking insects. 6.57mg/cm2 had 99% repellency against C. lectularius in treated sur- face after 9 hours. Further studies are recom- mended to determine the protection time and repellency of oregano essential oil using EC99 on animal and human hosts. Acknowledgements The authors wish to thank Dr Mohammad J Arthropod-Borne Dis, December 2018, 12(4): 387–397 M Sharififard et al.: Chemical Composition … 395 http://jad.tums.ac.ir Published Online: December 25, 2018 Mahmoodi Sourestani for helping in interpre- tation of GC-MS results. This article is part of Ismaeil Alizadeh MSPH thesis and it was financially supported by and conducted in In- fectious and Tropical Diseases Research Cen- ter, Health Research Institute, Ahvaz Jun- dishapur University of Medical Sciences (AJUMS). (Project No. OG-95102). The authors declare that there is no con- flict of interest. References 1. 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