J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 105 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Repellency Effect of Essential Oils of some Native Plants and Synthetic Repellents against Human Flea, Pulex irritans (Siphonaptera: Pulicidae) *Mohammad Bagher Ghavami 1, Fahimeh Poorrastgoo 1, Behrooz Taghiloo 2, Jamshid Mohammadi 1 1Department of Medical Entomology and Vector Control, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran 2Zanjan Health Center, Zanjan University of Medical Sciences, Zanjan, Iran (Received 5 Jan 2016; accepted 7 Feb 2016) Abstract Background: Fleas are important vectors of human and animal disease, and control measures for protection against their bites and flea-borne diseases are necessary. Methods: The essential oils (EOs) of four native medicinal plants, Ziziphora tenuiore, Myrtus communis, Achillea wilhelmsii and Mentha piperita were isolated by hydrodistillation technique and analyzed by GC-MC. The repellent activity of EOs and synthetic compounds, DEET and permethrin, were assayed on human subjects against field col- lected fleas. The effective doses of 50% and 90% of EOs and synthetic compounds were estimated by probit analysis of dose and response regression line. Results: Analysis of EOs revealed about 19 major components. All oils were found to be more repellent (ED50 range= 208–955µ g cm-2) than DEET and permethrin (ED50 range= 27–182x 103µ g cm-2). Thyme and myrtle oils showed high repellent activities and among the total detected terpenes, thymol (36.26%) and α- pinene (32.5%) were the major components of those oils respectively. Conclusion: Low repellent potency of DEET and permethrin against fleas might be related to flea olfactory system and further molecular and electrophysiological studies are required to conceive new ideas for the discovery and de- velopment of the next generation of repellents. Based on high repellent activity of thyme and myrtle essential oils against Pulex irritans further studies should be staged to develop their appropriate effective formulations. Likewise, field trials should be carried out to evaluate the operational feasibility and dermal toxicity over a long period. Keywords: Pulex irritans, Ziziphora tenuiore, Myrtus communis, DEET, Repellents Introduction The human flea, Pulex irritans Linnaeus, is the most invasive species worldwide with great medical importance and intolerable nui- sance. The control of this vector has received considerable attention since ancient times. The most effective measure to control hu- man flea lies in adult stage using adulticides (Service 2012). Although application of syn- thetic insecticides can be effective, their in- tensive use has caused concerns regarding their impact on human and environmental health leading to development of resistance in the flea populations like other vectors (Hem- ingway and Ranson 2000). In addition to adulticides, personal protection including the use of repellents can decrease contact between human and fleas. N, N-Diethyl-3-methylben- zamide (DEET) has been considered as one of the most effective synthetic repellents against various groups of vectors and still re- mains as “the gold standard” among current- ly available insect repellents (Swal et al. 2014, Debboun et al. 2014 ). However, there has been no report concerning the repellency of DEET on human flea. In addition, there have been case reports of DEET toxicity especial- *Corresponding author: Dr Mohammad Bagher Ghavami, Email: Ghavami@zums.ac.ir J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 106 http://jad.tums.ac.ir Published Online: March 14, 2017 ly among children and the elderly (Qui 1998, Sudakin 2003). Moreover, genotoxic effects of this compound have been found previ- ously (Manikhan et al. 2012). In addition to DEET, permethrin, a synthetic pyrethroid, is characterized by a high level of potency against a wide range of vectors, rapid reac- tivity, excellent photostability, resistance to weathering, low mammalian toxicity (Yu 2014) and high potency in tick and insect bite prevention (Faulde et al. 2003, 2006). However, skin irritation, including itching, swelling, redness, mild burning and stinging may occur after treatment with permethrin. Moreover, arthropods show differential re- sponses to these products, indicating the pos- sibility of adaptation and emerging resistance or insensitivity (Klun et al. 2004, Stanczyk et al. 2010, Pellegrino et al. 2011, Sfara et al. 2011, Del Fabbro and Nazzi 2013). Thus, there is a growing demand to evaluate natural re- pellents to develop suitable agents for prophy- lactic treatment in integrated pest management. Plant essential oils (EOs) specially their monoterpen components; exhibit a variety of biological activities against a wide spectrum of insect pests. They can adversely affect the growth and reproduction rate, behavior trait of insect pests and act as contact insecticide, fumigant, repellent and antifeedent agent. These compounds have a low risk profile on the environment, mammals, and humans and might comprise natural alternatives for con- ventional insecticides (Bakkali et al. 2008, Koul et al. 2008). Fresh and dried shoots of myrtle, mint, thyme, yarrow and their EOs and extracts are widely used in cosmetic, food, gum, tooth- paste and pharmaceutical industries. These plants have also been commonly used in folk medicine for treatment of colds, pains and as spices and herbal tea as well (Naghibi et al. 2005, Bakkali et al. 2008, Saeidnia et al. 2011). Moreover, these plants proved to be toxic to different species of insects and ticks (Maia and Moore 2010, George et al. 2008, 2009, 2014, Pitarokili et al. 2011, Yahoobi- Ershadi et al. 2011, Tavassoli et al. 2011, Koc et al. 2012, Lupi et al. 2013). For human flea, however, little work has been done on EOs repellent activity. Moreo- ver, despite the great need in human health area for fleas prophylaxes by natural prod- ucts, the current knowledge concerning re- pellent action of EOs on human flea is very limited. Therefore, in the present study the chemical properties and components of some EOs were evaluated. Additionally a compar- ative study was conducted for detection of flea repellent activity of these Eos. Materials and Methods Plant materials The shoots of native medicinal plants, thyme (Ziziphora tenuiore), mint (Mentha piperita), yarrow (Achillea wilhelmsii) and myrtle (Myr- tus communis) were collected from different areas of Zanjan and Fars Provinces during May–Jun 2013 and 2014. Collected herbs were dried in shade for 8 days at room tem- perature until they became brittle. Air-dried samples were powdered in electric vegetable chopper and subjected to hydro distillation in a Clevenger type apparatus for about 3h. The extracted oils were dried over anhydrous sodium sulfate and refrigerated at 4 ˚C in dark glass bottles until the experiment. Gas Chromatography and GC mass analysis The gas chromatography (GC) analysis was carried out with an HP Series II 7890N in- strument equipped with flame ionization de- tector and HP-5-MS capillary column (30m× 0.25mm, 0.25µm film thickness). Injector and detector temperatures were set at 260 ˚C and 270 ˚C respectively. Thermal program of GC oven started with 60 ˚C for 4min, then rose to 60–225 ˚C at a rate of 3oc/min. Helium was the carrier gas at a flow rate of 1ml/min. Diluted samples (1:50 in diethyl ether) of 1µ l were injected manually in the split less mode. J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 107 http://jad.tums.ac.ir Published Online: March 14, 2017 Quantity analysis of EOs components was performed using the same condition with GC HP Series II equipped with HP GC 5975C mass selective detector in the electron im- pact mode 70eV. The oil components were identified by comparison of the retention time with those of authentic samples in mass spectra obtained from literature and the li- brary of the GC-MS system. Human subjects The study was conducted on 6 volunteers (four males and two females) who were aged between 28 to 50 years old. They were non- smokers, non alcoholics and non-allergic to insect bites or herbal oils. They also had no contact with lotions, perfumes, coils or per- fumed soaps on the days of assay. All vol- unteers signed an formal consent form after having received a full explanation of the test objectives, procedures and foreseeable risk to subject. The research protocol was ap- proved by the ethics committee of Zanjan University of Medical Sciences. Flea collection Alive samples of fleas were collected by human baits from animal shelters of Khoda- bandeh and Mahneshan districts of Zanjan Province from September 2013 to October 2014. Volunteers in white overalls and white ladies stockings, walked around the houses for 3–5 minutes and attracted fleas were suctioned by electrical aspirator and trans- ferred to laboratory in 2 liters Erlenmeyer flasks. Preparation of solutions Dilutions of each essential oil and DEET (Aldrich USA, D100951-500G, N, N-Diethyl- 3-methylbenzamide, lot# MKBH0428V, P Code: 1001075635, Density: o.97g/cm3) with grade reagent ethanol (ET) were made to stock in V/V at 85% concentration. From which five serial dilutions (42.75%, 21.37%, 10.68% and 5.34% concentrations) were pre- pared. Also a 20% permethrin (United Phos- phorus Ltd, Delhi India, with 96% active ingredient, 42:58 cis: trans ratio) solution was prepared by mixing 2.1g of permethrin with 10ml of grade reagent acetone and 5 serial dilutions (in acetone) were created. Repellency tests The bioassay was performed by the stand- ard protocols for testing mosquito and tick repellents (Dautel 2004, Bissinger 2010, Maia and Moore 2011, Krober et al. 2013, WHO 2013, Debboun 2014). About 60 unfed fleas were kept in laboratory condition (25±2 ˚C and 60% RH) and transferred to 20″×6″ clear cylinder glass vase. For testing, volun- teers used the left arm for treatment and the right for control. Five hundred µ l of test materials were applied to the treated area of left hand (from elbow to wrist, area approx- imately 500cm2) and allowed to dry for 5 minutes. The same area of right arm was applied with five hundred µ l of ET. After drying of treated area, both arms were cov- ered with white ladies stockings and volun- teers kept their hands (left as treatment and right as control) in glass jars about 3 minutes. The control hand was exposed before the treatment. Samples of fleas from different parts of arms (hand and wrist to elbow) were collected and recorded separately. These sam- ples were preserved in ET and observed under stereomicroscopy at 30×. Samples of Pulex irritans were recorded and other spe- cies of fleas were excluded from study. The percentage of repellency in each hand was calculated by the following formula: (Tc-Tb)/ (Tc+Tb)× 100, Where Tc= the number of fleas in hand of arm and Tb is the numbers in the area between elbow to wrist. The per- centage of repellency in treatment groups was corrected with control group by Abbot’s formula. The data were subjected to probit analysis and the median effective dose (ED50) and 95 % effective dose (ED90) with 95 confidence J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 108 http://jad.tums.ac.ir Published Online: March 14, 2017 interval exposed in microgram of repellent per square of centimeter of skin area were estimated. Significant differences between ef- fective dose values were determined by com- paring the confidence intervals of the values. Results Water distillation of the dried shoots of the examined plants furnished 0.8–1.2% (W/ W) of white to yellowish essential oils with strong pleasant odors were identified. Forty seven constituents were determined in the thyme essential oil representing 97.98% of total content. The main constituents were found to be thymol (36.26%), geraniol (11.61%), carvacrol (4.96%) and 1,8 cineol (3.26%) (Table 1). A total of twenty compounds were identi- fied in the oil of myrtle, representing around 99.15% of the oil totally. Alpha pinene (32.5%), 1,8 cineol (24.15), limonene (15.5%) linalool (10.6%) and linalyl acetate (5.5%) were the major constituents in the essential oil of Myrtus communis (Table 1) According to total 87.43% of the Achillea wilhelmsii oil, eighty constituents were iden- tified in the essential oil of this plant. The main components were camphen (19%), di- methyl heptatriene (10.15%), borneol (7.3%), artemisia alcohol (5.3%), menthen (4.56%) and 1,8 cineol (3.86%). There were fifty eight compounds in the oil of Mentha pipertta, comprising 98.26% of the total weight. Menthen (26.66%), piperi- ton (19.18%), piperiton oxide (19.07%), 1,8 cineol (8.5%) and trans caryophylen (3.29%) were the major compounds of essential oil of this plant (Table 1). During the study, 4529 samples of fleas were collected by human-bait catch. Human flea was the commonest species and 94.3% of samples were identified as Pulex irritans. The frequencies of other fleas were 3.8% and 1.9 % for Ctenocephalidescanis and C. felis re- spectively. The bioassay tests were carried out with 4271 samples of alive human flea. All tested essential oils in five concentra- tions showed differences in repellency with chemical compounds. The thyme essential oil provided highest repellency followed by myr- tle, yarrow and mint essential oils (Table 2). The EDs50 (with 95% confidence inter- vals) of thyme and myrtle essential oils were 229 (208–257) and 295 (269–338)μg/cm2 re- spectively. In these plants the ED90 (with 95% confidence intervals) of essential oils were 776 (660–933) and 1172 (988–1412) μg/cm2 respectively (Table 3). Yarrow essential oil showed moderate re- pellency against human flea. In this plant the ED50 and EDs90 (with 95% confidence inter- vals) of essential oil were 457 (371–630) and 2217 (1399–4673)μg/cm2 respectively. Mint essential oil provided slight repellency. The ED50 and ED90 (with 95% confidence inter- vals) of mint essential oil were 776 (660–855) and 5717 (3890–9120)µ g/cm2 respectively. Permethrin and DEET compounds showed low repellency in comparison with botanical essential oils. The ED50 of permethrin and DEET were estimated 7760 and 51000µ g/ cm2 respectively (Table 3). The related dose response lines and re- gression equations for essential oils and syn- thetic compounds are represented in Table 3 and Fig. 1. J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 109 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 1. Relative percentage of major components of essential oils of each plant extract No Compound name LRI (Min) Myrtus communis Achillea wilhelmsii Mentha piperita Ziziphora tenuiore 1 α - Pinene 6.37 32.5 2.59 0.73 0.71 2 Limonene 10.24 15.5 0 0 0 3 1,8 Cineol 10.36 24.15 3.86 8.6 3.16 4 γ - Tepinene 11.55 0 0.33 0.11 3.33 4 Artemisia alcohol 12.86 0 5.39 0 0 5 Linalooll 13.42 10.6 3.2 0.88 2.26 6 Dimethyhepta 14.67 0 10.15 0 0 7 Camphor 15.29 0 19 0.48 0.69 8 Borneol 16.38 0 7.30 0.35 3.26 9 Menthen 17.55 0 4.56 26.66 2.41 11 α -Terpinol 17.7 0 1.17 0.44 2.36 12 Linalyl acetate 21.14 5.5 0 0.25 0 13 Geraniol 21.45 0 0 0 11.61 14 Thymol 23.35 0 0.8 0.22 36.26 15 Carvacrol 23.57 0 0.26 0 7.98 16 Piperiton 24.99 0 0 19.18 0 17 Piperiton oxide 26.11 0 0 19.07 0 18 Geranyl acetate 26.68 0 0 0.40 4.72 19 Trans caryophylen 27.83 0 0 3.29 0 Total 88.25 57.76 76.8 78.75 Total Compound (No) 20 80 58 47 Total percentage 99.15 87.43 98.26 97.98 LRI*= Linear regression indices on HP-5 MS column relative to C9–C23 n alkanes , identification based on comparison of mass spectra obtained from the library of the GC-MS system and from literature. Table 2. Repellent activity (in percentage) of some essential oils and synthetic compounds against Pulex irritanson human volunteers Dosage (µ g/cm2) Study groups Ziziphora tenuiore Myrtus communis Achillea wilhelmsii Mentha piperita Permethrin DEET 50 3.2 3 3.1 3 2 2 100 17.7 17.5 12.5 15 7.2 5.2 200 48 36 22 20 8.2 6.2 400 74 63 46 32.5 9.3 7.3 800 87.5 80 72 48.4 12.4 10.4 1600 98 96 87 69 18.5 14.6 J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 110 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 1. Dose response lines for botanical and chemical repellent activity against Pulex irritanson human subjects. Table 3. Parameters of probit analysis on botanical and chemical compounds and their repellency against Pulex irritanson human volunteers Compound a b ED50 (95 % CE) (µ g/cm2) ED90 (95 % CE) (µ g/cm2) n X2 p Ziziphora tenuiore -5.78 2.44 229 (208–257) 776 (660–933) 828 101.7 <0.01 Myrtus communis -5.36 2.16 295 (269–338) 1172 (988– 1412) 895 99.6 <0.01 Achillea wilhelmsii -5.0 1.88 457 (371–630) 2217 (1399–4673) 169 102.1 <0.01 Mentha piperita -4.32 1.49 776 (660–955) 5717 (3890–9120) 822 68.7 <0.01 Permethrin -2.3 0.51 26.9 x 103 7.76x 106 362 15.0 <0.01 DEET -2.74 0.50 182 x 103 51 x 106 710 10.8 0.03 Discussion Evaluation of botanical compounds is es- sential to identify effective repellents for vec- tor control programming and protection of populations against vector nuisance and vector borne diseases. The insecticidal and repellent activities of various herbal derivatives have been demon- strated in many studies (Neiro et al. 2010, Bi- singer and Roe 2010, Lupi et al. 2013). However, only a few reports present the repellency of plant products against other groups of nuisance vectors especially fleas (George et al. 2009). In our investigation among examined plants, the essential oil of three plants, thyme, myrtle and yarrow, showed excellent to good repellency against all tested fleas. The oils of these plants were rich in volatile terpenes, thymol, α–pinene, 1, J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 111 http://jad.tums.ac.ir Published Online: March 14, 2017 8 cineol, camphor, limonene, linalool, and ge- raniol. In spite of effective repellency of these compounds, the major components of Mentha piperita, menthen and piperiton, showed low efficacy against fleas. Findings of previous studies have proved that, thymol and α–pinene are the most toxic compounds (Trabouli et al. 2002) and have effective repellent activity against mosqui- toes and ticks (Pandy et al. 2009, Jordan et al. 2012, Meng et al. 2015). High repellency of thyme and myrtle oils in our study might be associated with these components. Nev- ertheless, further laboratory and field studies are required to prove repellent efficacy of these compounds. Although the amounts of limonene, gera- niol, linalool and camphor in above-men- tioned essential oils were high in our study, they showed low potency of repellent activity against fleas. Furthermore, 1, 8 cineol which had low repellency against insects, showed different biological activities against mos- quitoes and ticks in previous studies (El- Seedi et al. 2012). In contrast to recent studies synthetic compounds, DEET and permethrin, which have shown effective repellency against mosqui- toes, could not repel human flea. Low effi- cacy of these compounds on Pulex irritans compared to other vectors might be related to their difference in mode of action (Dikens and Bohbot 2013, Daniel et al. 2014), mo- lecular characteristics of odorant binding pro- teins (Bohbot and Dikens 2012) and odorant receptors of fleas (Ditzen et al. 2008). This study encourages further electrophysiological and molecular studies on human flea to con- ceive new ideas for the discovery and de- velopment of the next generation of repel- lents. Thyme and myrtle oils showed high re- pellent activity among the tested oils against human flea on human subjects. The repellent effect of Ziziphora tenuiore essential oil showed the highest repellency (lowest ED50 value, 208µ g/cm2 and ED90 910µ g/cm 2). Ac- cording to our literature review, there is no published paper available on the repellent activity of Ziziphora tenuiore essential oil. Majority of published documents were in favor of thyme essential oil repellency against mosquitoes and only a few of them were conducted against ticks (Zhu et al. 2006, Park et al. 2012). In previous studies the lowest value of ED50 seen in laboratory study of Origanium majorans, a species of thyme group, against nymphs of Ixodesricinus was 15µ g/cm2 (Zhu et al. 2006). Thyme oil also showed highest repellency against lab- bred Aedes aegypti (ED50= 23µ g/cm 2) and Culex pipiens (ED50= 468µ g/cm 2). Our ED50 and ED90 estimated values for myrtle oil on human subject were 290 and 1172µg/cm2 respectively. In the laboratory study of Tavassoli et al. (2011) the ED50 and ED90 values for myrtle essential oil against lab-bred Anopheles stephensi were calculated as 110 and 540µ g/cm2 respectively. Moreover, in a laboratory study on animal model, using K and D apparatus against lab reared Phlebotomus papatasi, Yaghoobi-Ershadi et al. (2006) reported the ED50 and ED90 values of myrtle oil 114 and 671µ g/cm2 respec- tively. Interestingly, in our study the ED50 and ED90 values of myrtle oil are very close to these studies. Based on the repellent result against Pulex irritans we recommend Ziziphora tenuiore and Myrtus communis essential oils for further studies to develop appropriate effective for- mulations. Field trials should be carried out to evaluate the operational feasibility and der- mal toxicity over a long period. It is im- portant to determine whether widespread ap- plication of these repellents would produce an overall reduction of vector biting. Conclusion Based on our findings the two essential oils, thyme and myrtle, are preferable in terms of J Arthropod-Borne Dis, March 2017, 11(1): 105–115 MB Ghavami et al.: Repellency Effect of … 112 http://jad.tums.ac.ir Published Online: March 14, 2017 repellency effect against human flea. Further electrophysiological and molecular studies are recommended to identify the mode of action of these oils and nature of flea olfactory system in order to develop new generation of repellents. Measures to determine the appropriate formulation of recommended repellents and comprehensive field studies to evaluate their potency in bite reduction and possible ad- verse effects are necessary. Acknowledgment This work was financially supported by ZUMS. The authors would like to express gratitude to Dr Bighlari, chief of ZUMS and Dr Faghihzadeh, deputy of research and in- formation technology of ZUMS for their valuable help. We would like to thank staff of Zanjan, Mahneshan, and Khodabandeh, Health Centers for their kind collaborations. 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