J Arthropod-Borne Dis, June 2018, 12(2): 101–107 A Sanei-Dehkordi et al.: Essential Oil … 101 http://jad.tums.ac.ir Published Online: June 12, 2018 Original Article Essential Oil Composition and Larvicidal Evaluation of Platycladus orientalis against Two Mosquito Vectors, Anopheles stephensi and Culex pipiens Alireza Sanei-Dehkordi 1, 2, Sahereh Gholami 3, Mohammad Reza Abai 3, *Mohammad Mehdi Sedaghat 3 1Department of Medical Entomology and Vector Control, Faculty of Health, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 2Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 15 Jan 2017; accepted 11 Apr 2018) Abstract Background: Natural plant products as larvicides could be considered as desirable alternatives to synthetic chemi- cal insecticides for vector management. This study was undertaken to assess the mosquito larvicide activity of the essential oil from fresh leaves of Platycladus orientalis against two medically important species of mosquito vec- tors. Methods: Essential oil was extracted by hydrodistillation and analyzed with gas chromatography and mass spec- trometry (GC-MS). Fresh leaves of P. orientalis tree (500g) were collected in June 2014 from Tehran, Iran and was authenticated at the Department of Medical Entomology and Vector Control, School of Public Health, Tehran Uni- versity of Medical Sciences, Tehran, Iran. In addition, the larvicidal potential of oil was evaluated against late-3rd or young-4th instar larvae of Anopheles stephensi and Culex pipiens under laboratory condition. The mortality counts were made after 24h and LC50 and LC90 values were calculated. Results: Forty-six components in leaves of P. orientalis were identified. The major components were α-Pinene (20.17%), 3-Carene (14%) and Cedrol (9.51%). The LC50 values against An. stephensi and Cx. pipiens larvae were 11.67ppm and 18.60ppm after 24h, respectively. Conclusion: Platycladus orientalis oil could be considered as a natural larvicide for mosquito larval control. Keywords: Anopheles stephensi, Culex pipiens, Platycladus orientalis, Essential oil, Larvicide Introduction Mosquitoes are the most important group of arthropods with medical importance, which can transmit many pathogens and parasites cause important diseases such as malaria, dengue, yellow fever and filariasis throughout the world, except the Antarctic (1). Human malaria caused by protozoans (Plasmodium spp.) continues to be the most important vector- borne disease. It affects more than 91 tropi- cal countries, placing 3.2 billion people at risk as of 2015. Overall, 212 million cases of malaria and 429000 deaths were reported worldwide (2). This disease is transmitted only by the anopheline mosquitoes. There are more than 400 described species of Anopheles world- wide, from which about 40 species are im- portant vectors of human malaria (3). In Iran, malaria is transmitted by seven vector species, among these species, Anopheles ste- phensi is considered as a primary vector of malaria in the southern parts of Iran (4-6). Culex species are important vectors of hu- man pathogens worldwide including the eti- ologic agents of different forms of enceph- alitis, Rift valley fever, and lymphatic filari- *Corresponding author: Dr Mohammad Mehdi Sedaghat, E-mail: sedaghmm@tums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 101–107 A Sanei-Dehkordi et al.: Essential Oil … 102 http://jad.tums.ac.ir Published Online: June 12, 2018 asis. Culex pipiens with broad global distri- bution is considered as one of the most med- ically important vectors (1). There are several strategies applied for mosquito control, the usage of synthetic lar- vicides still remains as an effective method. The indiscriminate use of synthetic insecti- cides such as organophosphorus compounds have several adverse effects on environment, toxic effects on human beings and non-tar- get organism (specifically aquatic insects) and also may lead to the development of re- sistance in mosquito populations (7-9). The current trend has highlighted the search for new compounds for mosquito larval con- trol. From this viewpoint, botanical insecticides are promising since they are effective, environ- mentally friendly, degradability, non-toxic ef- fects on non-target organisms and often inex- pensive. Aromatic plants and their essential oils have been suggested as natural insecticides for pest control because they have few harmful effects on ecosystem structure and function (10). The insecticidal properties of plant vola- tile oils and extracts from a wide variety of plants have been assessed against the larvae of different species of mosquito (11-14). Platycladus orientalis (Linnaeus) Franco [synonym: Thuja orientalis Lineous, P. stricta Spach, Biota orientalis locally named as Sarv-e Khomrei or Noosh, is a monoecious and ev- ergreen tree belonging to the Labiatae fam- ily which grows wild in Korea, Japan, Chi- na, and Iran. This plant is cultivated as a common ornamental plant in different parts of Iran and other countries (15). Platycladus orientalis is used in Iranian traditional medicine as an astringent, stom- ach tonic, diuretic, tonic and antipyretic ef- fect (16). The essential oil of P. orientalis has been evaluated for antimicrobial activity (17), antifungal activity (18, 19), cytotoxico- logic activity (20), molluscicidal activity (21) and insecticide activity (22, 23). The main purpose of this study was to analyze the essential oils by GC-MS in order to identify the constituents and larvicidal activ- ity of the leaf essential oil of P. orientalis. Materials and Methods Plant materials Fresh leaves of P. orientalis tree (500g) were collected in June 2014 from Tehran, Iran (51º 23’E, 35º 42’N, elevation: 1242m). The plant was identified and authenticated at Department of Medical Entomology and Vec- tor Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Essential oil isolation The fresh leaves of P. orientalis were cut into small pieces and subjected to hydrodis- tillation using Clevenger-type apparatus (Mod- el: British Pharmacopoeia, manufactured by Pyrexfan Company, Iran and mantle model EM manufactured by Bibby Scientific Com- pany, United Kingdom). The essential oil was dried over anhydrous sodium sulfate and the purified oil transferred into amber-colored vials at +4 °C until further use. Gas chromatography/mass spectrometry The oil analysis was carried out using GC- MS. The GC apparatus was Agilent technol- ogy (HP) 6890 system, capillary column of HP-1MS (Fused silica) (30m× 0.25mm, film thickness 0.25μm). The oven temperature program was initiated at 40 °C, held for 1 min then raised up to 230 °C, rising to 250 °C at 5 °C/min. The carrier gas used was he- lium at a flow rate of 1ml/min. The detector and injector temperatures were 260 °C and 230 °C, respectively. GC/MS analysis was conducted on an HP 6890 GC system cou- pled with a 5973 network mass selective detector with a capillary column the same as above, carrier gas helium with flow rate 1 ml/min. A sample of 1μL was injected in the split mode with split ratio 1:50. For GC–MS detection, an electron ionization system, with ionization energy of 70eV, was used, injec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 101–107 A Sanei-Dehkordi et al.: Essential Oil … 103 http://jad.tums.ac.ir Published Online: June 12, 2018 tor and oven temperature programmed was identical to GC. The Retention indices (RI) were calculated for all volatile constituents using a homologous series of n-alkanes (C8– C24) on the HP-1MS column. Quantification was performed using percentage peak area calculations and the identification of indi- vidual compartments was done using the Wiley 7n.1 GC-MS library, NIST (National Institute of Standards and Technology) and those published in the literature (24-26). Mosquito rearing The larvae of An. stephensi and Cx. pipiens were obtained from the anopheline insectary of Department of Medical Entomology, Teh- ran University Medical Sciences. The con- tinuous breeding colony of mosquito was main- tained at 27 °C with 12:12 L/D photoperiod in 65±5% RH. Larvae of An. stephensi were avail- able for the mosquito larvicidal experiments. Bioassays and larval mortality Bioassay tests were performed according to the standard method recommended by WHO (27). In brief, the essential oil first dissolved in absolute ethanol. A 400ml glass beaker was used for each experiment or control. The late- 3rd or young-4th instar larvae of An. stephensi and Cx. pipiens were exposed to 5, 10, 20, 40 and 80ppm of essential oil in water, in the con- trol beakers only solvent (absolute ethanol) was dissolved into the water. Mortality was counted after 24h. When necessary, mortality was ad- justed for control deaths using Abbott’s for- mula (28). The lethal concentrations of 50% and 90% mortality (LC50 and LC90) were cal- culated using Probit analysis (29). Results Yields and chemical constituents of essential oil The yield of essential oil was 0.5% (w/v) based on fresh weight. The essential oil was yellowish with a distinct sharp odor. Forty-six constituents in the essential oil of P. orientalis were identified corresponding to 97.88% of the total oil (Table 1). The major components of P. orientalis oil were identified as α-Pinene (20.17%), 3-Carene (14%) and Cedrol (9.51%). Other minor constituents were found to be β- Thujene (7.85%), Terpinolene (6.56%) and α- Terpinyl acetate (4.38%). Mosquito larvicidal activity of essential oil The larvicidal activity of leaf oil from P. orientalis against An. stephensi and Cx. pipiens under laboratory conditions are shown in Table 2. Among the five concentrations test- ed, the dosage of 80ppm could induce more than 90% mortality in both the species. The mortality rates in the control groups were lower than 5% in all concentrations. The LC50 and LC90 values against An. stephensi and Cx. pipiens larvae were 11.51, 67.81 ppm and 18.60, 127.24ppm after 24h, re- spectively. Among different concentrations tested, there were no significant differences in larval mortality between the two species (P> 0.05). In regression line a positive cor- relation was observed between the leaf oil concentrations and mortality rates (Fig. 1). Table 1. Chemical constituents of leaf essential oil from Platycladus orientalis Constituentsa RIb Composition% Tricyclene 922 0.26 α-Pinene 938 20.17 α-Fenchene 950 1.71 β-Thujene 966 7.85 β-Pinene 979 3.88 3-Carene 998 14.00 2-Carene 1006 0.77 D-sylvestrene 1024 0.50 Limonene 1032 2.74 β-Ocimene 1038 0.14 α-Ocimene 1041 0.08 γ-Terpinene 1056 1.73 cis-Thujane-4-ol 1062 0.15 Terpinolene 1083 6.56 1,3,8-p-Menthatriene 1110 0.05 cis-p-Menth-2-en-1-ol 1119 0.28 (4E,6Z)-allo-Ocimene 1130 0.02 Borneol 1164 0.06 Terpinene-4-ol 1176 2.95 α-Terpineol 1191 0.40 cis-Piperitol 1199 0.18 Fenchyl acetate 1225 0.06 cis-Geraniol 1228 0.01 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 101–107 A Sanei-Dehkordi et al.: Essential Oil … 104 http://jad.tums.ac.ir Published Online: June 12, 2018 Citral 1270 0.03 L-bornyl acetate 1282 1.89 α-Terpinyl acetate 1346 4.38 Nerol acetate 1366 0.42 Geraniol acetate 1383 0.57 β-Elemene 1389 0.82 α-Cedrene 1410 1.23 Caryophyllene 1424 4.32 gamma-Elemene 1433 0.63 α-Caryophyllene 1455 3.34 Germacrene D 1478 2.47 β-Bisabolene 1507 0.11 δ-Cadinene 1525 0.38 Elemol 1548 0.94 Nerolidol 1566 0.05 Caryophyllene oxide 1581 0.08 Cedrol 1602 9.51 γ-Eudesmol 1634 0.75 Cedryl acetate 1760 0.42 8,15-Pimaradiene 1890 0.02 Hexadecanoic acid 1971 0.02 Pimara-7,15-dien-3-ol 2250 0.78 Totarol 2301 0.20 Total 97.88 aCompounds listed in order of elution from a HP-1 MS column bRetention Indices as determined on HP-1MS using the homologous series of n-alkanes (C8–C24) Fig. 1. Probit regression line of Anopheles stephensi and Culex pipiens exposed to different interval concentrations of essential oil from fresh leaves of Platycladus orientalis Table 2. Parameters of probit regression lines of Platycladus orientalis oil against the larvae of Anopheles ste- phensi and Culex pipiens Species A B±SE LC50, 95% C.I. LC90, 95% C.I. χ 2 (df) P-value An. stephensi -1.77 1.66±0.159 11.51 67.81 5.9 (3) 0.05 Cx. pipiens -1.95 1.53±0.397 18.60 127.24 20.9 (3) <0.05 A: y-intercept, B: The slope of the line, SE: Standard error, LC50, 95% CI: Lethal concentration causing 50% mortality and its 95% confidence interval, LC90, 95% CI: Lethal concentration causing 90% mortality and its 95% confidence interval χ 2= heterogeneity about the regression line, df: degree of freedom p= represent heterogeneity in the population of tested Discussion The yield of oil obtained of P. orientalis leaves was 0.5% (w/v). This amount is sim- ilar to recent study (17) but relatively higher than other studies (22, 25,30, 31). According to our results, α-Pinene (20.17%), 3-Carene (14%) and Cedrol (9.51%) are known as the main compounds of the oil. Similar to those previously reported (17, 26, 30, 31), the ma- jor constituents were α-pinene, 3-carene and cedrol. However, there were differences in the amount of the main components. The differ- ences in the number of compositions in the above-mentioned studies may be due to the collection time and geographic factors (26). Essential oils obtained from plants have been studied as a natural compound poten- tially used as an alternative to common syn- thetic insecticides. Earlier authors reported Table 1. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 101–107 A Sanei-Dehkordi et al.: Essential Oil … 105 http://jad.tums.ac.ir Published Online: June 12, 2018 that the larvicidal activity of essential oils of various aromatic plants with LC50 values ranging from 24.27 to 105.4ppm and 20.61 to 311.2ppm against the larvae of An. ste- phensi and Cx. pipiens, respectively (12, 31- 37). The leaf oil of P. orientalis was very ef- fective against An. stephensi and Cx. pipiens with LC50 values of 11.51 and 18.60ppm af- ter 24h, respectively, which were much low- er than those of the plants studied earlier. In the present investigation, the dosage of 80ppm was sufficient to cause 100% larval mortality against the larvae of both species after 24h. Similarly, the toxicity of T. orien- talis and Chamaecyparis obtusa oils were tested against 4th-instar larvae of Aedes ae- gypti and Cx. pipiens after 24h and they re- ported 100% larval mortality when treated with 400ppm of both oils (23). According to proposed categories of lar- vicidal activity of plant essential oils against mosquito larvae, essential oil of P. orientalis can be considered as active (38) to very ac- tive (39) plant. Conclusion Essential oils of P. orientalis are prom- ising in mosquito control. These findings could be useful in search for newer, safer, and more effective natural larvicidal com- pounds against disease-vector mosquitoes. Further studies must be conducted to de- scribe the mode of action of each constituent separately also its effects on non-target or- ganisms. Acknowledgments This work was supported by a grant from the Center for Environmental Health Re- search, Tehran University of Medical Sci- ences, Tehran, Iran, Project No. 88-01-46- 8661. 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