J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 85 http://jad.tums.ac.ir Published Online: March 18, 2018 Original Article Larvicidal Activity of Bunium persicum Essential Oil and Extract against Malaria Vector, Anopheles stephensi Hassan Vatandoost 1, 2, Arezoo Rustaie 3, Zeynab Talaeian 3, Mohammad Reza Abai 1, Fatemeh Moradkhani 3, Mahdi Vazirian 3, Abbas Hadjiakhoondi 3, Mohammad Reza Shams-Ardekani 3, 4, *Mahnaz Khanavi 3, 5 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Department of Pharmacognosy, Faculty of Pharmacy and Persian Medicine and Pharmacy Research Centre, Tehran University of Medical Sciences, Tehran, Iran 4Department of Traditional Pharmacy, School of Traditional Medicine, Tehran University of Medical Sciences, Tehran, Iran 5Faculty of Land and Food Systems, University of British Columbia, Vancouver, Canada (Received 21 Sep 2016; accepted 12 Dec 2017) Abstract Background: Malaria, a mosquito-transmitted disease, is still a major human health problem all over the world. Lar- viciding is a component of comprehensive control program to overcome the disease. Negative aspects of synthetic insecticides application, such as environmental safety concerns, have favored use of natural insecticides. Methods: Larvicidal activity of essential oil, extracts and fractions of a wild grown and a cultivated type of Bunium persicum fruits against malaria vector Anopheles stephensi was assessed according to the method described by WHO. Results: Bunium persicum showed remarkable potency against An. stephensi larvae. LC50 values for essential oil, total extract, petroleum ether fraction and methanol fraction were 27.4284, 64.9933, 85.9933 and 255.7486ppm for wild type, and 21.3823, 63.2580, 62.7814 and 152.6357ppm for cultivated one. Conclusion: The results of this study suggest B. persicum as a valuable source of natural insecticides against malaria vector Anopheles stephensi. Keywords: Anopheles stephensi, Bunium persicum, Larvicidal activity, Extract, Essential oil Introduction Despite progresses made over the past dec- ades to decline the mortality rate of malaria all over the world, it is still prevalent in some tropical countries and areas with about 200 million affected cases in 2013. Vector con- trol interventions have had substantial con- tribution on the recent reduction in global malaria burden. Larviciding, with the aim of adult vector density reduction, as an auxiliary to core interventions, is helpful especially in urban regions, where breeding of vectors take places in permanent or semi-permanent aquat- ic habitats (1). The mosquito Anopheles ste- phensi is one of the six main vectors of hu- man malaria in southern parts of Iran (2). Larvicidal potentials of some herbal extracts and essential oils on An. stephensi larvae have been investigated previously (3-5). Bunium persicum is a perennial plant be- longing to Apiaceae family, growing wild in Iran (6). The fruit of B. persicum is used as spice, antiseptic and carminative agent (7). Sev- eral studies have analyzed essential oil compo- sition of the fruits and mostly reported γ-ter- *Corresponding author: Dr Mahnaz Khanavi, E- mail: khanavim@tums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 86 http://jad.tums.ac.ir Published Online: March 18, 2018 pinene, cuminaldehyde and ρ-cymene as main components (8-10). Kaempferol, caffeic and p- coumaric acid have been isolated from polar fraction of the fruits as major antioxidant con- stituents (11) but according to our knowledge no other comprehensive study has been orga- nized to identify other phytochemicals in the extract. Overexploitation and unscientific har- vesting of B. persicum as well as climate changes, has threatened its existence in wild (12). Cultivation of endangered species could preserve their genetic resources (13). In re- cent years, B. persicum is cultivated in lim- ited areas in Iran especially in Khorasan Raza- vi Province. As a part of our ongoing studies on larvicidal activity of plants extracts and essential oils against An. stephensi (4, 5, 14- 20), in the present study, we have studied larvicidal activity of the essential oil, extract and fractions from B. persicum fruits against late third instar larvae of An. stephensi. Moreover, we have compared the activities of a wild and a cultivated type. Materials and Methods Plant material The fruit of wild B. persicum was pur- chased from Kerman, and cultivated type was supplied from agricultural research fields of Ferdowsi University of Mashhad (2013). The samples were authenticated at the Herbarium of Faculty of Pharmacy, Teh- ran University of Medical Sciences, where voucher specimens were deposited (PMP- 649 and PMP-689). Essential oil preparation 100g powdered fruits of cultivated and wild B. persicum were subjected to hydrodistillation for 3 hours using Clevenger type apparatus. The obtained essential oils were dried over an- hydrous sodium sulfate and kept in refrigera- tor until needed. Extraction and fractionation 250g dried and powdered fruits from both samples were separately extracted with meth anol (5× 1.5L) to afford total methanol extracts. The solvent was removed under re- duced pressure by rotary evaporator at 40 °C, and subsequently lyophilized by freeze dryer at -40 ºC for 24h (Lyotrap Ultra, LTE Scientific Ltd., Oldham, UK). Fractionation of total extracts was performed with suffi- cient volumes of petroleum ether, ethyl ace- tate and methanol. The fractions were then concentrated to dryness by rotary evaporation. Larval mortality bioassay Anopheles stephensi larvae (Bandar Abbas strain) were supplied by the Department of Medical Entomology, Tehran University of Medical Sciences. The mosquito colony was maintained under a constant insectarium condition at 27 ºC and 75–85% relative hu- midity with 12:12 light and dark photoperi- od. Late third and early fourth instars larvae were used for experiments. Larvicidal activity of total extracts, frac- tions and essential oils were evaluated accord- ing to the procedure recommended by WHO (21). The larvae were exposed to different concentrations of samples for 24 hours. Tests were carried out in four replicates. One ml of solvents (DMSO for essential oil and petroleum ether fraction, DMSO2: water 3 for total extract and ethanol for methanol fraction) were added separately into control bakers. Mortality was scored 24 hours post exposure. Analysis method The mortality percentages were calculated and corrected relative to the associated con- trols using Abbott’s formula (22). The concen- tration-mortality data were subjected to Pro- bit analysis (23) and lethal concentrations (LC50 and LC90) were determined with 95% confidence intervals from the regression lines. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 87 http://jad.tums.ac.ir Published Online: March 18, 2018 Results Hydro distillation of wild and cultivated B. persicum fruits yielded 2.5% and 2.25% (w/w) essential oil respectively. Both essential oils had a lot of commonalities in composition. γ-Terpinene (30.77% and 27.57%), cuminaldehyde (20.49% and 21.1%), ρ-cy- mene (20.1% and 18.32%) and γ-terpinen-7- al (8.29% and 7.84%) constituted main com- ponents in the wild and cultivated oils re- spectively (24). The results of larvicidal ac- tivity of essential oils, total extracts, petrole- um ether and methanol extracts against An. stephensi under insectary condition are pre sented in table 1 and plotted in Figs. 1 to 4. All tested samples showed significant anti- larval effect against the malaria vector An. stephensi, of which, the essential oils from cultivated and wild types with LC50 values of 21.3823ppm and 27.4284ppm were the strong- est samples and methanol fractions with LC50 values of 152.6357ppm and 255.7486 ppm exhibited least larvicidal activity among the samples. Comparison of lethal concentration values of efficient tested samples reveals there is no difference in efficacy of them be- tween wild and cultivated types. Fig. 1. Comparison of lethal concentrations (LC50) of cultivated and wild types of Bunium persicum essential oils against larvae of Anopheles stephensi http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 88 http://jad.tums.ac.ir Published Online: March 18, 2018 Table 1. Probit regression line parameters of essential oil, total extract, petroleum ether and methanol fraction of wild and cultivated Bunium persicum fruits against Anopheles stephensi Specimen Intercept Slope ± SE LC50 95% CI LC90 95% CI χ2 P W EO -4.1233 2.8670 ± 0.471 27.4284 19.7868-35.0421 76.7752 56.3038-139.1275 41.682 <0.05 C EO -4.8215 3.6251 ± 0.486 21.3823 13.6913-25.9482 48.2608 38.6334-68.5159 33.107 <0.05 W T -3.9100 2.1568 ± 0.270 64.9933 44.8917-89.5814 255.3195 170.9457-498.5673 16.725 <0.05 C T -4.6503 2.5819 ± 0.181 63.2580 55.8062-71.3261 198.3795 167.9464-243.6008 10.718 <0.05 W PE -5.0602 2.6158 ± 0.502 85.9933 47.2631-130.9756 265.7116 166.2611-869.8822 28.442 <0.05 C PE -4.7365 2.6346 ± 0.186 62.7814 55.4789-70.6893 192.4364 163.2242-235.7975 12.880 <0.05 W M -6.6009 2.7414 ± 0.365 255.7486 159.3871-405.0692 750.4194 459.8467-2245.3156 26.381 <0.05 C M -9.6475 4.4181 ± 1.524 152.6357 94.5358-262.7553 297.6718 202.8848-6675.4919 18.475 <0.05 W: wild, C: cultivated, EO: essential oil, T: total extract, PE: petroleum ether fraction, M: metha- nol fraction, SE: standard error, LC50: lethal concentration to cause 50% mortality in population, LC90: lethal concentration to cause 90% mortality in population, CI: confidence interval, χ 2: heter- ogeneity about the regression line. Fig. 2. Comparison of lethal concentrations (LC50) of cultivated and wild types of Bunium persicum total extracts against against larvae of Anopheles stephensi http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 89 http://jad.tums.ac.ir Published Online: March 18, 2018 Fig. 3. Comparison of lethal concentrations (LC50) of cultivated and wild types of Bunium persicum petroleum ether fraction against against larvae of Anopheles stephensi Fig. 4. Comparison of lethal concentrations (LC50) of cultivated and wild types of Bunium persicum methanol frac- tion against larvae of Anopheles stephensi http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 90 http://jad.tums.ac.ir Published Online: March 18, 2018 Discussion Many researchers have already studied lar- vicidal potentials of plant derived compounds, extracts and essential oils against various in- sects, with the aim of finding active phyto- chemicals to replace synthetic insecticides. High cost of various commercial insecticides beside their food and environmental safety concerns, toxicity problems and increasing resistance rates have made their utilization undesirable (25, 26). Anti-larval activity of essential oil from a wild grown B. persicum against An. stephensi and Culex pipiens has been previously reported with LC50 values of 27.72 and 20.61ppm respectively (27). Ac- cording to the results of our study, the essen- tial oil, methanol total extract and petroleum ether fraction of both wild and cultivated samples had significant larvicidal activity against late third and early fourth instar lar- vae of An. stephensi. The larvicidal potential of γ-terpinene, cuminaldehyde and ρ-cymene, main constituents of both wild and cultivated type B. persicum fruits, against various insect larvae has been previously proved in several experiments. γ-Terpinene has shown potent larvicidal activity with LC50 value of 29.21 ppm against Anopheles anthropophagus (28) and 30.7 and 29.8ppm against Aedes aegypti and Aedes albopictus respectively (29). Zah- ran and Abdelgaleil (30) documented tox- icity of cumin aldehyde on Culex pipiens lar- vae, which was more stronger than other test- ed monoterpenes in that experiment, with LC50 values of 38.9 and 21.4ppm for 24 and 48h exposures respectively. Anti-larval potential of ρ-cymene, the other main constituent, to- wards A. aegypti and Ae. albopticus has also been demonstrated (LC50= 19.2 and 46.7ppm) (29). Higher lethal effect of the petroleum ether fraction in comparison to the methanol frac- tion, suggests higher potency of non-polar com- ponents than polar phenolics towards An. ste- phensi larvae. LC50 value of 85.9933 and 62.7814ppm for petroleum ether fraction from wild and cultivated types makes it suitable choice for further studies to isolate the active principles. Anti-larval activity of efficient sam- ples from cultivated type was comparable to those from wild grown, so it can be conclud- ed that cultivation of B. persicum has not affected chemical constituents’ biosynthesis or concentration, which are responsible for lar- vicidal activity of the fruit. Conclusion The extract and fractions from B. persicum fruits, ie, petroleum ether fraction and total ex- tract, beside the essential oil, have shown significant larvicidal effects on An. stephensi, and can be a great candidate to develop an eco- friendly insecticide to combat malaria vector breeding. More precise investigation will re- quire revealing phytochemical composition of extract. Since cultivated type showed com- parable results as wild grown, cultivation of B. persicum, as a solution to preserve its wild resources, is highly recommended. There are several studies on larvicidal activities of different plants against malaria vectors in Iran (16, 31-46). We recommend formulation of plant extract which have the lowest LC50 for field evaluation. Acknowledgment This project was supported by the deputy of research at Tehran University of Medical Sciences (TUMS) (grant number 26183) and was a part of Dr Z Talaeian, Pharmacology department. References 1. WHO (2013) Larval Source Management: a supplementary measure for malaria vec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 91 http://jad.tums.ac.ir Published Online: March 18, 2018 tor control: an operational manual. Ge- neva: World Health Organization. p. 116. 2. Soleimani-Ahmadi M, Vatandoost H, Hanafi- Bojd AA, Zare M, Safari R, Mojahedi A, Poorahmad-Garbandi F (2013) Envi- ronmental characteristics of anopheline mosquito larval habitats in a malaria en- demic area in Iran. Asian Pac J Trop Med. 6: 510–515. 3. Saxena R, Harshan V, Saxena A, Sukuma- ran P, Sharma M, Kumar ML (1993) Larvicidal and chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi. J Am Mosq Con- trol Assoc. 9: 84–87. 4. Hadjiakhoondi A, Vatandoost H, Jamshidi A, Amiri EB (2003) Chemical constitu- ents of efficacy of Cymbopogon olivieri (Boiss) Bar essential oil against malaria vector, Anopheles stepensi. Daru. 11: 125–128. 5. Khanavi M, Vatandoost H, Dehghani NK, Sanei-Dehkordi A, Sedaghat MM, Hadji- akhoondi A, Hadjiakhoondi F (2013) Lar- vicidal activities of some Iranian native plants against the main malaria vector, Anopheles stephensi. Acta Med Iranica. 51: 141–147. 6. Mozaffarian VA (2006) A Dictionary of Ira- nian Plant Names. Tehran: Farhang Moaser. pp. 198–515. 7. Amin G (2005) Popular Medicinal Plants of Iran. Vice chancellorship of Research, Tehran University of Medical Scienc- es, Tehran, Iran, p. 54. 8. Baser K, Oezek T, Abduganiev B, Abdul- laev U, Aripov KN (1997) Composition of the essential oil of Bunium persicum (Boiss.) B. Fedtsch. from Tajikistan. J Essent Oil Res. 9: 597–598. 9. Foroumadi A, Asadipour A, Arabpour F, Amanzadeh Y (2002) Composition of the essential oil of Bunium persicum (Boiss.) B. Fedtsch. from Iran. J Essent Oil Res. 14: 161–162. 10. Jahansooz F, Sefidkon F, Najafi A, Ebrahim- zadeh H, Najafi MS (2012) Compari- son of essential oils of Bunium persicum (Boiss.) populations grown in Iran, Paki- stan and India. J Essent Oil Bear Plants. 15: 761–765. 11. Sharififar F, Yassa N, Mozaffarian V (2010) Bioactivity of major components from the seeds of Bunium persicum (Boiss.) Fedtch. Pak J Pharm Sci. 23: 300–304. 12. Saeidnejad AH, Kafi M, Khazaei HR, Pes- sarakli M (2013) Effects of drought stress on quantitative and qualitative yield and antioxidative activity of Bunium persi- cum. Turk J Botany. 37: 930–939. 13. WHO, IUCN, WWF (1993) Guidelines on the conservation of medicinal plants. p. 120. 14. Hadjiakhoondi A, Vatandoost H, Abou- sa-ber H, Khanavai M, Abdi L (2008a) Chemical composition of the essential oil of Tagetes minuta L and its effect on Anopheles stephensi larvae in Iran. J Med Plants. 7(26): 33–39. 15. Vatandoost H, Khazani A, Rafinejad J, Khoobdel M, Kebriai-Zadeh A, Abai MR, Hanafi-Bojd AA, Akhavan AA, Abtahi SM, Rafi F (2008) Comparative efficacy of Neem and dimethyl phthalate (DMP) against malaria vector, Anophe- les stephensi (Diptera: Culicidae). Asia Pac J Trop Med. 1(3): 1–6. 16. Shahi M, Hanafi-Bojd A, Iranshahi M, Vatandoost H, Hanafi-Bojd M (2010) Larvicidal efficacy of latex and extract of Calotropis procera (Gentianales: As- clepiadaceae) against Culex quinquefas- ciatus and Anopheles stephensi (Diptera: Culicidae). J Vector Borne Dis. 47: 185– 188. 17. Khanavi M, Bagheri-Toulabi P, Abai MR, Sadat N, Hadjiakhoondi F, Hadjiakhoon- di A, Vatandoost H (2011b) Larvicidal activity of marine algae, Sargassums wartzii and Chondriada syphylla, against malaria vector, Anopheles stephensi. J http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 92 http://jad.tums.ac.ir Published Online: March 18, 2018 Vector Borne Dis. 48: 241–244. 18. Vatandoost H, Sanei-Dehkordi A, Sadeghi SMT, Davari B, Karimian F, Abai MR, Sedaghat MM (2012) Identification of chemical constituents and larvicidal ac- tivity of Kelussia odoratissima Mozaf- farian essential oil against two mosqui- to vectors Anopheles stephensi and Cu- lex pipiens (Diptera: Culicidae). Exp Parasitol. 132(4): 470–474. 19. Khanavi M, Fallah A, Vatandoost H, Sed- aghat M, Abai MR, Hadjiakhoondi A (2012) Larvicidal activity of essential oil and methanol extract of Nepeta men- thoides against malaria vector, Anophe- les stephensi. Asia Pac J Trop Med. 5 (12): 962–965. 20. Torabi Pour H, Shayeghi M, Vatandoost H, Abai MR (2016) Study on larvicidal effects of essential oils of three Iranian native plants against larvae of Anopheles stephensi (Liston). Vector Biol J. 1: 2. 21. WHO (1981) Instructions for determining the susceptibility or resistance of mos- quito larvae to insecticides. Geneva: WHO/VBC. p. 6. 22. Abbott W (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol. 18: 265–267. 23. Finney DJ (1971) Probit Analysis. 3d Ed. Cambridge University Press. Cambridge, p. 333. 24. Rustaie A, Keshvari R, Samadi N, Kha- lighi-Sigaroodi F, Shams Ardekani MR, Khanavi M (2016) Essential oil com- position and antimicrobial activity of the oil and extracts of Bunium per- sicum (Boiss.) B. Fedtsch., wild and cultivated fruits. Pharm. 22: 296–301. 25. Ghosh A, Chowdhury N, Chandra G (2012) Plant extracts as potential mosquito lar- vicides. Indian J Med Res. 135(5): 581– 598. 26. Shaalan EAS, Canyon D, Younes MWF, Abdel-Wahab H, Mansour AH (2005) A review of botanical phytochemicals with mosquitocidal potential. Environ Int. 31: 1149–1166. 27. Sanei-Dehkordi A, Vatandoost H, Abaei MR, Davari B, Sedaghat MM (2016) Chemical composition and larvicidal ac- tivity of Bunium persicum essential oil against two important mosquitoes vec- tors. J Essent Oil Bear Pl. 19: 349–357. 28. Zhu L, Tian YJ (2011) Chemical compo- sition and larvicidal effects of essential oil of Blumea martiniana against Anoph- eles anthropophagus Asia Pac J Trop Med. 4: 371–374. 29. Cheng SS, Huang CG, Chen YJ, Yu JJ, Chen WJ, Chang ST (2009) Chemical compositions and larvicidal activities of leaf essential oils from two euca- lyptus species. Bioresour Technol. 100: 452–456. 30. Zahran HEDM, Abdelgaleil SAM (2011) Insecticidal and developmental inhibi- tory properties of monoterpenes on Cu- lex pipiens L. (Diptera: Culicidae). J Asi Pac Entomol. 14: 46–51. 31. Hajiakjoondi A, Aghel N, Xamaniza- dehnadgar N, Vatandoost H (2000) Chemical and biological study of Men- tha spicata L. essential oil from Iran. Daru. 8(1 and 2): 19–21. 32. Hadjiakhoondi A, Vatandoost H, Khanavai M, Abai MR, Karami M (2005) Bio- chemical investigation of different ex- tracts and larvicidal activity of Tagetes minuta L. on Anopheles stephensi larvae. Iran J Pharmaceutical Sci. 1: 81–84. 33. Hadjiakhoondi A, Sadeghipour-Roodsari HR, Vatandoost H, Khanavi M, Abaee M, Vosoughi M, Kazemi M (2006a) Fat- ty acid composition and toxicity of Me- lia azedarach L. fruits against malaria vector Anopheles stephensi. Iran J Phar- maceutical Sci. 2: 97–102. 34. Hadjiakhoondi A, Vatandoost H, Khanavi M, Sadeghipour-Roodsari HR, Vosoughi M, KazemiM,Abai MR (2006b) Chem- ical composition and toxicity of Melia http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 85–93 H Vatandoost et al.: Larvicidal Activity of … 93 http://jad.tums.ac.ir Published Online: March 18, 2018 azedarach L. against malaria vector Anophels stephensi. Iran J Pharmaceu- tical Sci. 2: 97–102. 35. Hadjiakhoondi A, Vatandoost H, Kha- navi M, Abousaber M, Abdi M (2008b) Chemical components and efficacy of fresh and dry Tagetes minuta L. against Anopheles stephensi. J Med Plants. 26 (7): 33–39. 36. Oshaghi MA, Ghalandari R, Vatandoost H, Anopheles stephensi on human volun- teers. J Entomol Zool Stud. 3(2): 343– 347. 37. Mozaffari E, AbaiMR, Khanavi M, Vatan- doost H, SedaghatMM,Sanei-Dehkordi A, Moridnia A, Saber-Navaei M, Rafi F (2014) Chemical composition, larvi- cidal and repellent properties of Cionu- raerecta (L.) Griseb. Against malaria vec- tor, Anopheles stephensi Liston (Diptera: Culicidae) under laboratory conditions. J Arthropod Borne Dis. 8(2): 147–155. 38. Hosseini SA, Bazrafkan S, Vatandoost H, Abaei MR, Ahmadi MS, Tavassoli M, Shayeghi M (2014) The insecticidal ef- fect of diatomaceous earth against adults and nymphs of Blattella germanica. Asia Pac J Trop Biomed. 4(Suppl 1): S228– S232. 39. Pirmohammadi M, Shayeghi M, Vatan- doost H, Abaei MR, Mohammadi A, Bagheri A, Khoobdel M, Hasan Bakh-shi H, Pirmohammadi M, Tavassoli M (2016) Chemical composition and re- pellent activity of Achillea vermiculata and Satureja hortensis against Anophe- les stephensi. J Arthropod Borne Dis. 10 (2): 201–210. 40. Shayeghi M, Kamalinejad M, Tourabi- Khaledi H, Abolhassani M, Hashemza- deh M (2003) Repellent effect of ex- tracts and essential oil of Citrus limon (Rutaceae) and Melissa officinalis (Labi- atae) against main malaria vector, Anoph- eles stephensi (Diptera: Culicidae) in Iran. Iran J Public Health. 32(4): 47–52. 41. Vatandoost H, Moinvaziri M (2004) Lar- vicidal activity of neem extract (Aza- dirachta indica) against mosquito larvae in Iran. Eastern Mediterr Health J. 10 (4/5): 1–9. 42. Sedaghat M, Sanei-Dehkhordi AR, Khanavi M, Abai MR, Hadjiiakhondi A, Moh- tarami F, Vatandoost H (2010) Phyto- chemistry and larvicidal activity of Eu- calyptus camaldulensis against malaria vector, Anopheles stephensi. Asia Pac J Trop Med. 3(11): 841–845. 43. Sedaghat MM, SaneiDehkordi AR, Kha- navi M, Abai, MR, Mohtarami F, Vatan- doost H (2011) Chemical composition and larvicidal activity of essential oil of Cupressus Arizona E.L. Greene against malaria vector Anopheles stephensi Lis- ton (Diptera: Culicidae). Pharmacognosy Res. 3 (2): 135–139. 44. Khanavi M, Rajabi A, Behzad M, Hadjiakhoondi A, Vatandoost H, Abai MR (2011a) Larvicidal activity of Cen- taurea bruguierana ssp. Belangerana against Anopheles stephensi Larvae. Iran J Pharm Res. 10(4): 829–833. 45. Tavassoli M, ShayeghiM, Abai MR, Vatan- doost H, Khoobdel M, Salari M, Gha- deri M, Rafi F (2011) Repellency ef- fects of essential oils of Myrtle (Myrtus communis L.), Marigold (Calendula of- ficinalis L.) compared with DEET against Anopheles stephensi on human volun- teers. Iran J Arthropod Borne Dis. 5(2): 10–22. 46. Tavassoli M, Shayeghi M, Abai MR, Vatandoost H, Khoobdel M, Salari M, Ghaderi A, Rafi F (2015) Repellency effects of picaridin and DEET against Anopheles stephensi on human volun- teers. J Entomol Zool Stud. 3(2): 343– 347. http://jad.tums.ac.ir/