J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 201 http://jad.tums.ac.ir Published Online: January 05, 2016 Original Article Chemical Composition and Repellent Activity of Achillea vermiculata and Satureja hortensis against Anopheles stephensi Masoumeh Pirmohammadi 1, *Mansoureh Shayeghi 1, *Hassan Vatandoost 1,2, *Mohammad Reza Abaei 1, Ali Mohammadi 1, Akbar Bagheri 1, Mehdi Khoobdel 3, Hasan Bakhshi 1, Maryam Pirmohammadi 4, Maryam Tavassoli 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Iran 2Instituet for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 4Kashan University, Kashan, Iran (Received 3 Aug 2014; accepted 17 Jan 2015) Abstract Background: One of the best ways to control the malaria disease and to be protected human against Anopheles mos- quito biting is the use of repellents. Throughout repellents, herbal ones may be an appropriate and safe source for protection. Methods: Chemical constituents of Achillea vermiculata and Satoreja hortensis were determined by using gas chromatography-mass spectrometry. Efficacy and the protection time of these plants were assessed on Anopheles stephensi under the laboratory condition. Results: The mean assessed protection time and efficacy for A. vermiculata was 2.16 and 3.16 hours respectively and the obtained ED50 and ED90 for this plant was 5.67 and 63 µ l/cm 2 respectively. The figured for S. hortensis was 4.16 and 5 hours respectively. ED50 and ED90 for this plant were 5.63 and 45.75µl/cm2 respectively. Conclusion: Results of investigation showed that S. hortensis plant has an acceptable protection time, therefore, this plant could be considered as a good herbal repellent against anopheles mosquitoes. Keywords: Achillea vermiculata, Satureja hortensis, Anopheles stephensi, Repellency, Protection time Introduction Mosquito borne diseases affect human societies by reduction in labor productivity especially in tropical and subtropical coun- tries. The important point is this fact that all countries all over the world have a problem with insect borne diseases (Govindarajan et al. 2011). Anopheles mosquitoes are bloodsucking insects, responsible for transmission of ma- laria, filariasis and arboviruses (Service 1980). There are 33 currently recognized Anopheles species including sibling, biological forms and zygotypes, seven of these species have an important role in malaria transmission in Iran. Among these species, An. stephensi is consid- ered as a primary vector of malaria in south- ern parts of the country (Sedaghat et al. 2003, Vatandoost et al. 2004, Sedaghat et al. 2005). According to the WHO report, a total of 627000 people die due to malaria (WHO 2013). Malaria is caused by Plasmodium parasite. Malaria is one of the most important diseases which the parasite is transmitted by female Anopheles genus (WHO 2010). There are several methods for malaria vector control. *Corresponding authors: Dr Mansoureh Shayeghi, E-mail: mansorehshayeghi@yahoo.com, Dr Hassan Vatandoost, E -mail: hvatandoost1@yahoo.com, Dr Mohammad Reza Abaei, E-mail: abaimr@tums.ac.ir J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 202 http://jad.tums.ac.ir Published Online: January 05, 2016 Synthetic insecticides which are generally used have side-effects on human, animal health, and the environment. The side-effects of synthetic organophosphorus compounds on fish and other organisms in the environ- ment are being increasingly reported. A lot of attention is being paid to natural products in vector control as they are environmentally safe, degradable and target-specific. Recent studies have demonstrated that use of repel- lents is one of the effective ways to control the disease and to avoid Anopheles bites (Vatandoost et al. 2008). DEET is slightly yellow oil. It is the most common active ingredient in insect repellents. It is intended to be applied to the skin or to clothing, and provides protection against mos- quitoes and many other biting insects. DEET was developed during World War II. The findings bring evidence that, DEET has side effects, so it has proposed to use alternative repellents for protection (Karunamoorthi et al. 2010). Some prefer to use natural insect repellent products. Repellents of plant origin do not pose hazards of toxicity to human and domestic animals and are easily biodegrada- ble. Natural products are safe for human when compared to that of synthetic compounds (Fradin 1998). The effect of some plant origin essential oils have been tested in Iran (Oshaghi et al. 2003, Yaghoobi-Ershadi et al. 2006, Vatandoost and Hanafi-bojd 2008, Tavassoli et al. 2011, Mozaffari et al. 2014 ). Achillea vermiculata is a flowering plant in the family Asteraceae with a height of 10– 30 cm. It is native to temperate regions of the Northern Hemisphere in Asia, Europe, and North America. It is an erect herbaceous perennial plant. The leaves have varying de- grees of hairiness. The leaves are almost feathery (Mozaffarian 2012). Satureja hortensis has lilac tubular flow- ers. It grows to around 30 to 60 cm in height and has very slender, bronze-green leaves. This plant belongs to order: Lamiales and family: Lamiaceae. It is used in traditional medicine as a botanical treatment (Mozaffarian 2012). This study was conducted to evaluate the repellent properties of two plants A. vermiculata and S. essential oil against An. stephensi in laboratory condition on animal model and also to determine chemical com- positions in their essential oils. Materials and Methods Mosquitoes rearing Established colony of susceptible strain of An. stephensi obtained from the Insectary of School of Public Health, Tehran Univer- sity of Medical Sciences, Tehran, Iran. Mos- quitoes were reared and maintained at 28±2 °C and 65±5% relative humidity (RH) under a 16:8 (L: D) photoperiod. Larvae were fed on a diet of fish food and water lettuce. The adults were maintained in screen cages and fed with 10% aqueous sucrose solution as a source of energy and guinea pigs as blood- feeding female mosquitoes for maturing the eggs. Starved 5 to 8 days old females were used for the repellency tests. The sucrose solution was picking up from the cage, 12 hour before starting the experiments. Collection, identification and extraction of plants Fresh flowers and leaves flowers of A. vermiculata and S. hortensis were collected from Armand and Sheyda district which are located in Chaharmahal and Bakhtiari Prov- ince in south-west of Iran in June 2013 (Fig.1,2). They were rapidly transported to the School of Public Health, Tehran Univer- sity of Medical Sciences. Achillea vermiculata was collected from natural habitat in Armand district at coordinate 31° 39.428′E 50° 46.659′N, 1136 meters above sea level. Satureja hortensis was collected from natural habitat in Sheyda district in Ben at coordinate 32° 37.206′E 50° 42.434′N, 2219 meters above sea level. J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 203 http://jad.tums.ac.ir Published Online: January 05, 2016 The plant A. vermiculata was identified by experts in Department of Plant Sciences, Tehran University. Also S. hortensis was iden- tified by experts in Ecotoxicology and her- barium laboratory, School of Public Health, Tehran University of Medical Sciences. The flowers and leaves of A. vermiculata and S. hortensis were dried at room temperature under good ventilation. About 525 gr of dried A. vermiculata and 420 gr of dried S. hortensis were chopped into small pieces using a knife mill. The essential oil was ex- tracted from the plants using a Clevenger- type water steam distillation apparatus. It took about 4 hours for extraction of the es- sential oils. For extraction of essential oil of each plant we used Sulphate Sodium Anhydr. The distilled essential oils were stored in a refrigerator at 4 °C until being used in the experiments. The composition of the volatile constituents was established by gas chromatography-mass spectrometry. Test method All series of the experiments of effective dose and protection time were carried out in laboratory condition. We tested these plants on white rabbits. The white rabbits (O. cuni- culus) (laboratory reared albino male aged 6–8 months) were used to determine both protection time and effective dosage in in- sectary at the School of Public Health. In this investigation, the 50% concentration of essential oils was used for protection time test. For this purpose essential oils were di- luted by absolute ethanol. Then for estimat- ing the protection time of plants essential oils against An. stephensi the back of male rabbits by 52cm2 were shaved in the next stage, 80µ l of 50% essential oils of two plants by sampler on the shaved back of male rabbits were applied. After 5 minutes the rabbits were placed in a box which is de- signed for the test. Then it is placed in box containing the rabbit in a cage at dimension of 53×53×53 cm containing 150 starved 5–8 days mosquitoes. After 3 minutes of biting and probing records, we brought out the cage and we tested again 30 minutes later. These tests continued until two successive bites. This time is called protection time. We continued these tests until 10 bites. This time is called failure time (Pitasawat et al. 2003). The procedure for determination of effec- tive dosages of the repellents was adopted by the standard method of American Society for Testing and Material (ASTM 2000). The testing kit was made of plexiglas cube at dimension of 4×5×18cm having four rectangular holes 4×3cm. Before starting the test for determination of effective dosage, the abdomen skins of rabbits were cleaned with alcohol and the kit was fixed on the abdomen. Each of 4 adjacent cells of kit was provid- ed with 5 female 5–8 days mosquitoes that randomly selected from a cage containing 150 starved mosquitoes. Circles were drawn on the rabbit's skin. The drawn circles on the abdomen skin’s of hold rabbit were treated with 50μl of essential oil diluted with abso- lute ethanol at 6.6, 13.2, 26.4 and 52.8µ l for A. vermiculata and 3.3, 6.6, 13.2, 26.4 and 52.8µ l for S. hortensis microliter with 4 rep- etitions. The same dilutions were applied on 3 holes because of prevention of contamina- tion as well as the absolute ethanol was ap- plied in remaining control circle. We used 5 mosquitoes for each hole. The treated circles were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on the treated skin. The counts of probing and biting were recorded for 5 minutes. After each test, the mosquitoes were transferred to netted cups and the mortality of mosquitoes was recorded after 24 hours. The ED50 and ED90 values and regression parameters were analyzed using probit 79 programs and the regression lines were plotted in Microsoft Excel 2007. Plants essential oils analysis Chemical composition of A. vermiculata J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 204 http://jad.tums.ac.ir Published Online: January 05, 2016 and S. hortensis was analyzed using an Agilent 7890–5975 gas chromatography- mass spectrometer. With a HP- 5MS (5% Phenyl Methyl Silox) capillary column (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization de- tector. The GC was programmed at 50 °C for 0.5 min and then increased at 5 °C/min to 280 °C, and finally held with an isothermal for 3min. The injector temperature was 280 °C. The flow rate of the carrier gas was 1ml/min. The identification of compounds was performed by comparing their retention times and mass spec- tra with mass spectra from Wiley library. Results Essential oil volumes By the use of Clevenger-type water steam distillation, about 1849µl of essential oil of 525gr of dried A. vermiculata flowers was extracted. Also about 4480µ l of essential oil of 420gr of dried S. hortensis leaves extracted. GC-mass analysis One microliter of each essential oil was injected to GC-mass. A total of 40 compounds were identified in flowers of A. vermiculata. (E)-β-damascenone with 27.4, (E)-2-hexenal with 8, eugenol with 6 and geranyl acetone with 6 percent were the major components (Table 1). We just found a repellent compo- nent “camphene” of all identified components by researchers until now in this plant with 0.7%. Also we identified 23 components in the leaves of S. hortensis. B-oplopenone with 57, trans-carvone oxide with 15.13 and thymol methyl ether with 13 percent were the major components (Table 2). Protection time The protection time of A. vermiculata es- sential oil against An. stephensi on animal subject provided 2.0–2.5 hours range with a mean of 2.16 hours protection and a failure time of 3–3.5 hours range with a mean of 3.16 hours. Also the protection time of S. hortensis essential oil provided 4–4.5 hours range with a mean of 4.16 hours protection and a failure time of about 5 hours (Table 3). Significant differences of protection time and failure time between A. vermiculata and S. hortensis repellents were observed by ANOVA (Games-Howel), P< 0.05. Effective dose The ED50 and ED90 values of A. vermiculata essential oil were 5.67 and 63 µ l/cm2 with confidence interval ranged, 2.25-8.68 and 38.21–198.07 µ l/cm2 respec- tively (Table 4). The ED50 and ED90 values of S. hortensis essential oil were 5.63 and 45.73 µ l/cm2 with confidence interval ranged, 3.83-7.43 and 30.92–86.55 µ l/cm2 respectively (Table 4). We did not observe any significant differ- ences between ED50 and ED90 of S. hortensis and A. vermiculata by T-test and P> 0.001 analysis. Fig. 1. The plant Achillea vermiculata in its natural habitat, Armand district in Chaharmahal and Bakhtiari Province, south-western of Iran (original) J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 205 http://jad.tums.ac.ir Published Online: January 05, 2016 Table 1. Chemical constituents of flower essential oil from Achillea vermiculata NO Compound Composi- tion% RI 1 isovaleric acid 0.2 833 2 (E)-2-hexenal 8 854 3 Tricyclene 0.1 921 4 Limonene 0.3 1029 5 Methybenzoate 0.5 1093 6 Linalool 3.1 1108 7 α-campholenal 2.8 1131 8 Camphene 0.7 1156 9 Pinocarvone 0.7 1164 10 cis-piperitol 0.7 1194 11 Verbenone 0.8 1208 12 trans-carveol 4 1222 13 cis-carveol 3.7 1233 14 Geraniol 1 1258 15 2E,4E-decadienal 3.1 1313 16 Eugenol 6 1359 17 (E)-β-damascenone 27.4 1382 18 cis-a-bergarnotene 5.6 1416 19 trans-a-bergamotene 3 1433 20 geranyl acetone 6 1452 21 allo-aromadendrene 0.8 1462 22 gamma-gurjunene 0.3 1473 23 a-muurolene 0.6 1499 24 a-cadinene 1.2 1537 25 Spathulenol 0.7 1579 26 b-oplopenone 0.4 1601 27 humulene epoxide II 0.3 1609 28 silphiperfol-6-en-5-one 0.6 1623 29 1-epi-a-eudesmol 2 1660 30 8-cedren-13-ol 0.3 1694 31 Xanthorrizol 0.3 1751 32 8-a-acetoxyelemol 2 1788 33 Nootkatone 0.4 1802 34 Flourensiadiol 1.8 1864 35 Hexadecanol 1.7 1881 36 methyl hexadecanoate 1.9 1911 37 methyl hexadecanoate 1.8 1925 38 methyl hexadecanoate 2.2 1941 39 methyl hexadecanoate 2.6 1957 40 Heneicosane 0.25 2057 99.55 Table 2. Chemical constituents of leaf essential oil from Satureja hortensis NO Compound Composition% RI 1 isovaleric acid 0.06 831 2 (Z)-3-hexenol 2.1 856 3 trans-sabinene hy- drate 0.3 1095 4 Linalool 1 1109 5 α-campholenal 0.6 1132 6 Borneol 1 1171 7 trans-carveol 2 1221 8 thymol methyl ether 13 1237 9 trans-carvone oxide 15.1 1277 10 Undecanal 0.4 1306 11 β-caroyophyllene 3 1424 12 b-oplopenone 57 1600 13 10-epi-gamma- eudesmol 0.3 1620 14 Hinesol 0.6 1637 15 8-cedren-13-ol 1 1704 16 Oplopanone 0.2 1726 17 8-a-acetoxyelemol 0.3 1781 18 Flourensiadiol 1.04 1867 19 methyl hexadecanoate 0.12 1910 20 methyl hexadecanoate 0.2 1923 21 methyl hexadecanoate 0.2 1935 22 Heneicosane 0.1 2097 23 n-docosane 0.2 2213 99.82 Fig. 2. The plant Satureja hortensis in its natural habitat, Sheyda district in Chaharmahal and Bakhtiari Province, south-western of Iran (original) J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 206 http://jad.tums.ac.ir Published Online: January 05, 2016 Fig. 3. Dose-response lines for two botanical repellents against Anopheles stephensi on animal model Table 3. Protection time and failure time of Achillea vermiculata and Satureja hortensis against An. stephensi on animal subject in laboratory condition Failure time (hour)Protection time (hour)DistrictSpecies (plants) MeanRangeMeanRange 3.163–3.52.162–2.5LordeganA. vermiculata 55–54.164–4.5BenS. hortensis Table 4. Effective dose of Achillea vermiculata and Satureja hortensis essential oils against Anopheles stephensi on animal subject in laboratory condition p-Valueχ2 table (df) χ2 (heterogeneity) ED90 (mg/cm2) ± 95% C.L. ED50 (mg/cm2) ± 95% C.L. b ± SEaplants 0.0122.3563 (38.21–198.07) 5.67 (2.25–8.68) 1.22±0.27-0.092A. vermiculata 0.0133.1345.75 (30.92–86.55) 5.63 (3.83–7.43) 1.4±0.20-1.05S. hortensis Discussion Application of larvicides and repellents are generally accepted as they play an important role in control of the mosquitoes. The use of botanical essential oils as repellents against vectors of malaria disease including An. gambiae and A. stephensi has been tested suc- cessfully (Seyoum et al. 2002). In this study the components of A. vermiculata essential J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 207 http://jad.tums.ac.ir Published Online: January 05, 2016 oil were identified. (E)-β-damascenone with 27.4, (E)-2-hexenal with 8, eugenol with 6 and geranyl acetone with 6 percent were the major components. Totally 40 components were identified from this plant. In an in- vestigation by Ahmadi et al. (2011), on A. santolina, 29 components were identified from this plant which the major components were Camphor, Alpha-pinene, Camphene and 1,8 Cineole with 26.27, 10.14, 9.09 and 8.26 percent respectively. We found two same components “Camphene” and “Linalool” of A. vermiculata with A. santolina. Also we identified 23 components in the leaves of S. hortensis. B-oplopenone with 57, trans-carvone oxide with 15.13 and thymol methyl ether with 13 percent was the major components. In an investigation by Kamkar et al. (2013) (32 components were identified and reported from this plant which the major components belonged to γ- terpinene with 24.72%, thymol with 29.1% and carvacrol with 26.6%. In this investigation, we iden- tified some components which had been iden- tified in Kamkar et al. (2013) study. These components are: borneol, linalool and thymol. Also cis-sabinene hydrat was identified in Kamkar investigation, but our investigation revealed the presence of trans-sabinene hy- drate. In another investigation by Tajalli et al. (2012) on this plant, the major components were thymol, carvacrol, gamma-terpinene with 48.67, 8.96, 9.16 and 9.16 percent respec- tively. In our investigation, we found thymol and linalool which had been identified in Tajalli et al. (2012) study too. Our investiga- tion revealed that A. vermiculata essential oil can have 2.16 hours protection time and also its Failure time is 3.16 hours. Compared to A. vermiculata, S. hortensis could have 4.16 hours protection time and a Failure time of 5 hours which is about 2 times as much as A. vermiculata’s protection time and Failure time. Also we revealed an ED50 and ED90 effective dose of 5.67 and 63 µ l/cm2 respec- tively, while for S. hortensis they were 5.63 and 45.75 µ l/cm2 respectively. The repellency effect of essential oils of some plants has been studied in Iran. On a laboratory trial by Vatandoost et al. (2008), the repellency of neem tree’s essential oil against An. Stephensi in animal subject was determined. The ED50 and ED90 values of neem tree’s essential oils were calculated 0.159 and 1.388 mg /cm2 respectively. Also the protection time and effective dose of this plant calculated 31 minutes and 65 minutes respectively. The repellency effect of essential oils of both Myrtus communis and Calendula offi- cinalis had been reported against An. stephensi on human subject and the effective dose of these plants was 0.11 and 0.6 mg/ cm2 respectively. Also the protection time and Failure time for M. communi were 4.36 and 4.4 hours respectively. The protection time and Failure time for C. officinalis were 2.15 and 3.30 hours respectively (Tavassoli et al. 2011). In an investigation, the mean protection time of 50% essential oil of Cionura erecta (L) provided 2.28 hours protection against An. stephensi. The figures for ED50 and ED90 values were 10.12 and 23.01ppm re- spectively (Mozaffari et al. 2014). We estimate that the most protection time of mentioned investigations and our investi- gation, belongs to M. communi and S. hortensis with a protection time of 4.36 and 4.16 hours and failure time of 4.4 and 5 hours respectively. The weakest protection time and failure time belongs to neem plant by 31 minutes and 65 minutes respectively. There have been so many investigations about repellency effects of plants against mosquitoes all over the world until now (Moore et al. 2002, Rajkumar and Jeanesabn 2007, Mullai et al. 2008, Karunamoorthei et al. 2010, Shahi et al. 2010). Finally we recommend S. hortensis as a candidate for prodution of insect repellents because of its high protection against mos- J Arthropod-Borne Dis, June 2016, 10(2): 201–210 M Pirmohammadi et al.: Chemical Composition … 208 http://jad.tums.ac.ir Published Online: January 05, 2016 quitoes and also medicinal properties without any side effects. Although this plant did not show any significant differences of effective dose rate with A. vermiculata. We recom- mend doing more investigations on this plant. Conclusion According to the results it could be con- cluded that the plant is appropriate for the repellent formulation for mosquito control, although the field trail should be conducted in a malarious areas. Acknowledgments This study was funded and supported by Tehran University of Medical Sciences (TUMS), we are thankful to the staff of In- sectary of Culicidae, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences for the mass production of the mosquitoes for this study. We are thankful to Dr Salmaki, assistant professor from De- partment of Plant Sciences, Tehran Univer- sity for detection of A. vermiculata. 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