J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 60 Original Article Evaluation of Repellency Effect of Essential Oils of Satureja khuzestanica (Carvacrol), Myrtus communis (Myrtle), Lavendula officinalis and Salvia sclarea using Standard WHO Repellency Tests *Mohammad Hassan Kayedi 1, Ali Akbar Haghdoost 2, Ali Salehnia 3, Kiumars Khamisabadi 4 1Razi Herbal Medicines Research Center and Department of Parasitology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran 2Research Center for Modeling in Health, Kerman University of Medical Sciences, Kerman, Iran 3Khorraman Herbal Medicines Pharmaceutical Company, Khorramabad, Iran 4National Health Center, Tehran University of Medical Sciences, Tehran, Iran (Received 21 Feb 2012; accepted 30 Sep 2013) Abstract Background: Using special lotions and repellent sprays on skin is one of the effective methods to prevent Arthro- pods biting which was verified in this study. Methods: Essential oils of four plants (Satureja khuzestanica, Salvia sclarea, Lavendula officinalis and Myrtus communis) were separately extracted by Clevenger used hydro distillation method. Then separated solutions with 10%, 20% and 40% concentrations of essential oils of plants in 99.6 % ethanol were prepared. WHO guidelines for efficacy testing of mosquito repellents for human skin were used on different concentrations of essential oils of plants, traditional repellents (DEET, 50% and 33%) as positive control, and ethanol 99.6% and naked hands as nega- tive controls. Results: In negative control groups, the number of bits were comparable (P= 0.42) and had decreasing time trends (naked hands P= 0.011, ethanol P< 0.001). In all time points, minimum bites were observed in traditional repellents and it was significantly less than the other groups (P< 0.001). The time trend in the number of bites in the other groups was positive and showed minimum number of bites in time zero in all groups. We also found that the con- centration of repellents had association with the number of bites. The maximum and minimum numbers of bites were observed with 10% and 40% concentrations respectively in all groups. Conclusion: Essential oils of Salvia sclarea, Lavendula officinalis and Myrtus communis have repellency effect, even with 10% concentration of essential oils. Keywords: Malaria, repellents, Anopheles stephensi, essential oil, plants Introduction Using of special lotions and repellent sprays on skin is one of the effective methods to pre- vent Arthropods biting. However, research to find more effective repellents with minimum side effects is a very attractive research field for scientists with different backgrounds. As an example, DEET (N, N-diethyl-3-me- thyl benzamide) is one of the most effective repellents used worldwide for six decades (Katz et al. 2008). However, similar to other repellent, it might have irritant effects mainly on sensitive skins or other side effects (Antwi et al. 2008, Katz et al. 2008). In addition, the maximum efficacy of these substances varies between one up to twelve hours based on their ingredients and concentrations (Katz et al. 2008). Until now the environmental pro- tection agency’s has approved DEET, picaridin, MGK-326, MGK-264, IR 3535, oil of citron- ella, and oil of lemon eucalyptus as Insect *Corresponding author: Dr Mohammad Hassan Kayedi, E-mail: kayedi78@yahoo.co.uk http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 61 Repellent (IR) ingredients for application to the skin (Katz et al. 2008). Based on the above explanation, continu- ously search is needed to find more potent materials with minimum side effects and long- er durability. Among these studies, research on the repellency effect of essential oils and national products of plants has a very unique position. Satureja khuzestanica, Salvia sclarea, Lavendula officinalis and Myrtus communis grow in West of Iran, across Zagros moun- tains and other parts of the country. Insecti- cidal and larvicidal effects of different doses of essential oils of S. khuzestanica and M. communis have been reported (Uehleke and Brinkschulte-Freitas 1979, Traboulsi et al. 2002, Yang et al. 2004, Yi et al. 2006, Cetin et al. 2007, Martinez-Romero et al. 2007, Knio et al. 2008, Amer and Mehlhorn 2009). Other researchers have stated antimicrobial, anti- fungal and antiparasite effects of mentioned plants and essential oils of two other plants (L. officinalis and S. sclarea) that grow in Iran (Hayder et al. 2004, Shahidi-Bonjar 2004, Sonboli et al. 2006, Pinto et al. 2006, Ben Arfa et al. 2006, Appendino et al. 2006, Mahdi et al. 2006, Burt et al. 2007, Cristani et al. 2007). Repellency effect of essential oil of L. officinalis has been reported (Nazemi Rafi and Moharamipour 2008). Local people in some districts of Lorestan Province in West of Iran (Zagross mountains), use dried leafs of local plant “S. sclarea” inside their pillows. They believe that the plant protect them from bit- ing of insects during night (Ali Salehnia, per- sonal communication). Although scattered evidence is available about the repellency effects of the mentioned four substances, we did not find any studies that compare their effects using standard methodology. So, based on above explana- tions we evaluated repellency effect of es- sential oils of 4 mentioned plants using Stand- ard WHO Repellency Tests. Materials and Methods Extraction method L. officinalis (Ostoqudoos, local name, Fam- ily: Lamiaceae) is a decorative plant and is used for decorations of parks and gardens. We bought dried leafs and flowers of men- tioned plant from Hamedan City in West of Iran (latitudes 34.8000 and longitudes 48. 5167). M. communis (Moort, local name, Family: Myrtaceae) was collected around the Khorramabad City in West of Iran (latitudes 33.4841 and longitudes 48.3525). This plant grows in West of Iran. The name, species and family of selected parts of plants were con- firmed by academic staffs of Research Center of Agricultural Department, Lorestan Province (Shahla Ahmadi, personal communication). Leafs and flowers of L. officinalis and leafs of M. communis were dried and grind. Essential oils of plants were separately ex- tracted by Clevenger (model BP, British Phar- macopoeia, manufacturer Ashke Shisheh Com- pany, Iran and mantle model H610 manu- facturer Fater Company, Iran) used hydro distillation method at 60 ºC. Duration of ex- traction was 1 hour for each 0.5cc essential oil. Sodium solfate was used for dehydration (Stahl-Biskup and Saez 2002, Okoh et al. 2010, Gavahian et al. 2012). All above procedures were carried out in laboratories of Razi Herbal Medicines Re- search Center, Lorestan University of Medi- cal Sciences. We extracted respectively 3cc, 1.3cc and 0.5cc pure (about 100%) essential oils of 100gr of dried flowers and leaves of L. officinalis and dried leaves of M. communis. Essential oils of S. khuzestanica (carvacrol, Family: Lamiaceae), and S. sclarea (salvia, Family: Lamiaceae) were bought from Kho- raman Pharmaceutical Company, Khorram- abad, Iran. This company culture S. khuzes- tanica and extracts its essential oil (carvacrol) for medicine marketing. S. khuzestanica grows in West of Iran (South of Lorestan and North http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 62 of Khuzestan Provinces) and S. sclarea grows in West of Iran. The mentioned company ex- tracted respectively 3cc and 0.5cc pure (about 90% to 94%) essential oils of 100 gr of dried leaves of S. khuzestanica and S. sclarea (Ali Salehnia, personal communication). All essen- tial oils were kept in 4 ºC. Repellency tests WHO guidelines for efficacy testing of mosquito repellents for human skin were used (WHO 2009). Separate solutions with 10%, 20% and 40% concentrations of essen- tial oils of plants in 99.6% ethanol were pre- pared. Traditional repellents (DEET, 50% N, N-diethyl-3-methyl benzamide) and 33% N, N-diethyl-3-methyl benzamide and negative control (pure ethanol, 99.6%) were bought re- spectively from Nomad, Turnpike lane, Lon- don N8 OPX, Medical limited and School of Pharmacy, Shiraz University of Medical Sci- ences in Iran. The essential oils, traditional repellents as gold standard repellents (positive controls) and negative controls were rubbed on right and left forearms of four eligible volunteers from elbow to wrist and their hands were pro- tected by gloves to make it unattractive to mosquitoes. The whole forearm exposed in a mosquito cage (50cm× 50cm×50cm) with 50, 2 or 3 days old females of susceptible strain of An. Stephensi reared in insectarium, unfed and hungry. Hands were kept inside the cage for 45 seconds and numbers of bites were recorded. This procedure was repeated 3 times. Negative controls (naked hands and hands rubbed with pure ethanol) and positive controls (DEET, 50% N, N-diethyl-3-methyl benzamide and Shiraz 33% N, N-diethyl-3- methyl benzamide) were used in this experi- ments as well. All tests were repeated every hour up to five hours after the first tests. The used mosquitoes for tests changed in every test (WHO 2009). Repellency tests were started at 5.30AM and continued to 10.30AM. We used 3 cages of An. Stephensi per day each cage had 50 mos- quitoes. Tests were carried out in a half-dark room (27 ºC and 70% humidity) near insectary. The tests were carried out in Bandar Ab- bas Health Research site affiliated to Na- tional Institute of Health, Tehran University of Medical Sciences. This research has been approved by Ethical Committee of Lorestan University of Medical Sciences. Statistical Analysis With respect to the ethical considerations, we minimized the number of humans that take part in the project, and the first author of the paper personally participated in the trial. However, in order to maximize the sta- tistical power of our analysis and according to WHO guidelines for efficacy testing of mosquito repellents for human skin, we re- peated each trail three times, with six meas- urements over 5 hours. The data were computerized using SPSS version 11.5. The number of bites in each ex- periment was the main dependent variable. We checked the distribution of the number of bites in each concentration and each group by comparing the 95% confidence in- tervals of their skewness and kurtosis with their values in a normal distributed variable; since we did not find any significant differ- ence in these analyses, we used parametric tests to compare the number of bits groups and concentrations. We used repeated meas- ure ANOVA to assess the effect of time and also the type of repellents. In addition, the linear trends in the number of bites were computed using regression model. In all sta- tistical analysis, 0.05 was considered as sig- nificant level. Results The repellency effects of Nomad (DEET, 50% N, N-diethyl-3-methyl benzamide) and Shiraz (33% N, N-diethyl-3-methyl benzamide) in all tests were comparable up to 5 hours http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 63 (P= 0.571), so we decided to incorporate these two traditional groups to improve the power of statistical tests as gold standard positive control (DTA). Based on the results of few first tests with essential oil of S. khuzestanica, side effects were considerable, mainly the burning, reddening of skin, and irritation for several hours after rubbed on forearms, so essential oil of S. khuzestanica was dropped from repellency tests. Figure 1 shows the marginal means of the number of bites per session (adding together the results of different concentrations based on the results of multivariate analysis) of An. stephensi up to 5 hours classified by the type of repellents and control groups. Based on these findings, in negative control groups, the number of bits were comparable (P= 0.42) and had decreasing time trends (naked hands P= 0.011, ethanol P< 0.001), started from around 30 bites in time zero, reached to around four- teen in negative control group (naked hands), and six in another negative control group (ethanol, 99.6% ) after five hours. In all time points, minimum bites were observed in DTA group (less than 2 bites) and it was significantly less than the other groups (P< 0.001). The time trend in the num- ber of bites in the other groups was positive and showed minimum number of bites in time zero in all groups (less than 2 bites). How- ever, this trend in L. officinalis was more prominent (the average of bites in time five was 9.8, P= 0.001), while the corresponding num- bers in S. sclarea and M. communis were less than four (P= 0.04 and P= 0.011) respectively. The regression coefficients for the effect of time (which shows the average of changes in the number bites between two consecutive measures) were 0.29, 0.40, 0.58, and 0.20 for S. sclarea, M. communis, L. officinalis and DTA (as positive control) respectively. While these coefficients for negative controls (naked hands and pure ethanol) were -2.88 and -3.60. There were significant difference in the number of bites between negative controls and other repellents (P< 0.001). In time five, the difference between negative controls and M. communis was significant (P= 0.04). How- ever, the other groups had significant differ- ence only with negative control groups in time five (P< 0.05). We also found that the concentration of repellents had association with the number of bites (Fig. 2). The maximum and minimum numbers of bites were observed with 10% and 40% concentrations respectively in all groups. In Salvia and M. communis groups, only the difference between 10% and 40% concentra- tions were significant (P= 0.032 and P= 0.015 respectively). While in L. officinalis even the difference between the minimum and maxi- mum concentrations had borderline P-value (0.063). Fig. 1. The time trends in the number of bites per session classified by the type of repellents and control groups http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 64 Fig. 2. Comparison between numbers of bites in different concentrations classified by the type of repellents Discussion Our results showed that the essential oils of M. communis, L. officinalis and S. sclarea have repellency effects for An. stephensi. Alt- hough by time, their repellency effect de- creased but even after five hours, their effects were more than the effects in control groups. We found a sharp decreasing trend in the number of bites in negative control groups. This decreasing trend might be explained by this fact since we started our experiments very early morning and continued up to 10.30AM. However, this decreasing trend was not ob- served in the other groups which might be due to the attenuation in the repellency ef- fects of these substances. By time, the repellency effect of essential oils of plants was reduced but even after five hours still they had significant effect more or less comparable with the effect of DTA which is a known and standard repellent. Although the maximum effect was observed in samples with 40% concentration, even the effects of samples with 10% concentration were significant and comparable with higher concentrations. This means that for further studies 10% concentration can be used to as- sess the repellency effects of these substanc- es or even similar ones. These findings are consistent with a similar study carried out by Tavassoli et al. (2011). Although their meth- ods was different with our methods, they reported that repellency efficiency of 50% concentration of essential oil of M. communis (myrtle) was moderate and lower than DEET. Other researchers have studied repellency effects of other plants in Iran. Oshagi et al. (2003) evaluated repellency effect of extracts and essential oils of Citrus limon Burm, (lemon) and Melissa officinalis, (Balm) using An. stephensi in laboratory on animal and hu- man in comparison with DEET. They showed significant differences between oils and ex- tracts (P< 0.05) and reported that difference between DEET and lemon oil was not sig- nificant. They recommended lemon essential oil as an alternative to DEET. In another study Vatandoost and Hanafi-Bojd (2008) com- pared the repellency effect of 3 topical repel- lents (permethrin, DEET and neem tree ex- tract) against An. stephensi in Iran. They re- ported that:” Major heterogeneity of response was observed using DEET. Although neem was http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 65 the least effective agent, extracts of locally produced neem oil offer a promising repel- lent against mosquito biting”. Repellency effect of eucalyptus based plant has been shown by researchers in different tri- als in the Bolivian Amazon (Moore et al. 2002), China (Yang and Ma 2005) and Guinea Bissau (Palsson and Jaenson 1999). Ansari et al. (2005) reported that Pine oil (Pinus longifolia, Fam- ily: Pinaceae) which is used by people in In- dia for protection against mosquito bites pro- vide 100% protection against An. culicifacies up to 11 hours and 97% protection against Culex quinquefasciatus for nine hours. They recommended that aromatic oil of Citronella and Lemon eucalyptus may be promoted as a repellent. Two investigators reported repel- lency effect of neem tree extract from trials in Iran (Vatandoost and Hanafi-Bojd 2008) and Bolivian Amazon (Moore et al. 2002). Assessment of the repellency effects of essential oils of plants is a very attractive field for entomologists around the world (Palsson and Jaeson 1999, Curtis et al. 2002, Oshaghi et al. 2003, Seyoum et al. 2003, Omolo et al. 2004, Innocent et al. 2008, Vatandoost and Hanafi-Bojd 2008). Many researchers have carried out trials on repellency effect of different plants. In a study 41 plant extracts and 11 oil mixtures were evaluated using Aedes aegypti, An. stephensi and Culex quinquefasciatus on human volunteers. They reported Litsea (Litsea cubeba), Cajeput (Melaleuca leucadendron), Niaouli (Melaleuca quinquenervia), Violet (Viola odorata), and Catnip (Nepeta cataria) that have been most effective oils of plants with 100% repellency against three mentioned mosquitoes and up to 8 hours protection time (Amer and Mehlhorn 2006). Investigators reported re- pellency effect of six plant species that grow in the Kenyan coast (Odalo et al. 2005), three plant species that grow in Western Kenya (Seyoum et al. 2003) and six plant species that grow in the Kenya (Odalo et al. 2005). Their insecticidal and larvicidal effects have been documented, for example, the essential oils of S. khuzestanica and M. communis had significant larvicidal and insecticidal effects (Traboulsi et al. 2002, Amer and Mehlhorn 2006, Yic G et al. 2006, Cetin et al. 2007, Martinez-Romero et al. 2007). Nonetheless, their toxic effects might be a point of con- cern particularly after high dose of oral con- sumption (Uehleke and Brinkschulte-Freitas 1979). Jaenson et al. (2006) reported that: Lavan- dula angustifolia (lavender oil) and Pelargo- nium graveolens (geranium oil) have 100% repellencies against host-seeking nymphs of Ixodes ricinus when diluted to 30% in 1.2 propanediol. His results were similar to our results with L. officinalis. A study that was carried out in Iran by Nazemi and Mohara- mipour (2008), showed the repellency effect of L. officinalis on red flour beetle, Tribolium castaneum. They showed that after five hours the repellency effect was still considerable and significant, also they showed positive as- sociation between the concentration and re- pellency effect. These results were consistent with our results in the present study. Conclusion We found that the different concentra- tions (40%, 20% and 10%) of essential oils of S. sclarea, L. officinalis and M. communis had considerable repellency effect on An. Stephensi even with low concentration and after five hours. These findings might open windows for formulating new repellent with less side effect and longer durability. Acknowledgements The authors would like to thank the dep- uty of research affairs of Lorestan University of Medical Sciences, for funding the research, Dr Bahram Delfan, Lorestan University of Medical Sciences for his suggestions on herbs http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 60–68 MH Kayedi et al.: Evaluation of Repellency … 66 and their collaborators Abbas Pakari, Hassan Jawdan and Mossa Hatami in Bandar Abbas Health Research center affiliated to National Health center, Tehran University of Medical Sciences for their help in repellency tests and Marzieh Rashidi our collaborator in Razi Herb- al Medicines Research Center, Lorestan Uni- versity of Medical Sciences for her assistance in extraction and preparation of essential oils of plants and other collaborators for their as- sistance during experimental studies. The au- thors declare that there is no conflict of interest. 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