J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 577 http://jad.tums.ac.ir Published Online: October 04, 2016 Original Article Chemical Compositions of the Peel Essential Oil of Citrus aurantium and Its Natural Larvicidal Activity against the Malaria Vector Anopheles stephensi (Diptera: Culicidae) in Comparison with Citrus paradisi Alireza Sanei-Dehkordi 1,2, *Mohammad Mehdi Sedaghat 3, Hassan Vatandoost 3, Mo- hammad Reza Abai 3 1Department of Medical Entomology and Vector Control, Faculty of Health, Hormozgan Univer- sity of Medical Sciences, Bandar Abbas, Iran 2Social Determinants in Health Promotion Research Center, 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 29 Dec 2014; accepted 3 Nov 2015) Abstract Background: Recently, essential oils and extracts derived from plants have received much interest as potential bio- active agents against mosquito vectors. Methods: The essential oils extract from fresh peel of ripe fruit of Citrus aurantium and Citrus paradisi were tested against mosquito vector Anopheles stephensi (Diptera: Culicidae) under laboratory condition. Then chemical com- position of the essential oil of C. aurantium was analyzed using gas chromatography-mass spectrometry (GC–MS). Results: The essential oils obtained from C. aurantium, and C. paradisi showed good larviciding effect against An. stephensi with LC50 values 31.20 ppm and 35.71 ppm respectively. Clear dose response relationships were estab- lished with the highest dose of 80 ppm plant extract evoking almost 100% mortality. Twenty-one (98.62%) constitu- ents in the leaf oil were identified. The main constituent of the leaf oil was Dl-limonene (94.81). Conclusion: The results obtained from this study suggest that the limonene of peel essential oil of C. aurantium is promising as larvicide against An. stephensi larvae and could be useful in the search for new natural larvicidal com- pounds. Keywords: Citrus aurantium, Citrus paradisi, Essential oil, Larvicidal activity, Anopheles stephensi Introduction Mosquitoes are very significant vectors from the medical entomology's point of view. They are responsible for the transmission of many diseases to man and animals such as malaria, dengue, yellow fever, encephalitis or filariasis (Eldridge 2000). Human malaria, as a mosquito-borne disease, caused by para- sitic protozoa Plasmodium consider as the most important vector-borne disease, which is transmitted only by females of Anopheles mosquitoes (Lehane 1991). Malaria is still an endemic disease in certain foci located in south and southeast of Iran (Manouchehri et al. 1992, Sedaghat et al. 2003 a,b, Vatandoost et al. 2012). In this part of the country six anopheline mosquitoes including An. stephensi, are known as the main malaria vectors (Manouchehri et al. 1976, Sedaghat and Har- bach 2005). Anopheles (Cellia) stephensi Lis- ton 1901 is an important malaria vector with wide distribution in the Arabian Peninsula and the Indian subcontinent. It has also dis- tributed in in Khuzestan, Fars, Kerman, Hor- mozgan, Sistan va Baluchestan and southern *Corresponding author: Dr Mohammad Mehdi Sedaghat, E-mail: sedaghat@hotmail.co.uk J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 578 http://jad.tums.ac.ir Published Online: October 04, 2016 Kermanshah provinces in Iran (Manouchehri et al. 1976, Vatandoost et al. 2006, Hanafi et al. 2011). Several methods have been applied for con- trol of the vectors of the disease including using synthetic pesticides as larvicides or imagicides in the malaria control program. Organophosphate compounds are the most common chemical larvicides which are used in control of Anopheles. However, their tox- icity to fish and other non-target organisms and the environment are increased (Mittal et al. 1991, Pinkney et al. 1999). Also re- sistance of anopheles mosquitoes to these compounds has appeared in many areas (Vatandoost and Borhani 2004, Vatandoost et al. 2004). A lot of attention is being paid to the plant extracts or their essential oils as an alternative source of mosquito larval con- trol agents (Isman 2000, Sedaghat et al 2011a, b, Vatandoost et al. 2012). Citrus aurantium Linnaeus (Rutaceae) which called as bitter orange or marmalade orange is too sour, but the juice of ripe fruit is used as a condiment in Iran. The peel of C. aurantium is often used in marmalade and dried peel is used in different food and drinks. The flowers are used in tea and its essential oil is used in perfumes and orange- flower water, which is used to flavor sweets (Kiple and Ornelas 2000). The dried whole fruit or peel of the fruit is used in Asian and Western herbal medicine to treat digestive problems (Wichtl 1994). The hydrolate of the flowers has been used for treatment of mild depression, sedation and as a heart ton- ic for many years in Iran (Zargary 1986, Ayenechi 1991). The studies on biologic ef- fects of C. aurantium indicated potential mos- quito repellent, larvicidal and insecticidal activities of this plant (Cetin et al. 2006, Sumroiphon et al. 2006, Yoon et al. 2009). Citrus paradisi Macfadyen (Rutaceae) or grapefruit like other citrus fruits contains many phytochemicals which contribute to a healthy diet (Fellers et al. 1990). It is used in Persian traditional medicine to treat infected injuries, some digestive problems, cold and helps to lower cholesterol. Recent studies have shown the potential of C. paradise to favorably affect metabolic syndrome, lipid and sugar metabolism (Fujioka et al. 2006, Goldwasser et al. 2010, Ogura et al. 2011). Its seed extract has shown the antimicrobial properties against bacteria and fungi (von et al. 1999). The objective of this work was to study the effect of the peel essential oils of ripe fruits of C. aurantium and C. paradisi against fourth instar larvae of An. stephensi under la- boratory conditions and determine the chem- ical composition of C. aurantium essential oil. Materials and Methods Collection of plant materials Fresh plants samples of ripe fruit of C. aurantium and C. paradisi were collected in October 2012 from Babol, Iran (52º 41’E, º36 32’N, elevation: -5 m above sea level) and Jiroft (57º 45’E, 28º 37’N, elevation: 650 m above sea level) respectively. The plants were identified and authenticated and the voucher specimen was deposited at Vec- tor Biology Laboratory, Department of Med- ical Entomology, Tehran University of Med- ical Sciences, Iran. Extraction of essential oils The peel essential oils of fresh (50 g) of C. aurantium and C. paradisi were hydrodis- tilled using a Clevenger-like apparatus (Mod- el: British Pharmacopoeia, Manufactured by Pyrexfan Company, Iran and mantle model EM manufactured by Bibby Scientific Com- pany, United Kingdom) for 3 hours at 70 °C. The yields were averaged over four experi- ments and calculated according to fresh weight of the plant materials. The obtained oil was dried over anhydrous Na2SO4 and transferred into an airtight amber-colored vial at 4 °C for further experimentation by J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 579 http://jad.tums.ac.ir Published Online: October 04, 2016 gas chromatography–mass spectrometry (GC and GC-MS). GC and GC/MS analysis of essential oil GC analysis was carried out using an HP6890 gas chromatograph equipped with flame ionization detector and an Hp-1 capil- lary column (30 m× 0.25 mm I.D., film thick- ness 0.25 μm) and split ratio, 1:25. The GC settings were as follows: initial oven tem- perature was held at 40 °C for 1 min, rising to 250 °C at 5 °C/min. The injector tempera- ture was maintained at 250 °C. The detector temperature was at 230 °C. The carrier gas used was Helium at a flow rate of 1 ml/min. GC-MS was performed on Agilent Tech- nology 5973 mass selective detector con- nected with an HP 6890 gas chromatograph. The oil of C. aurantium was analyzed using an HP-1MS (Fused silica) with the same col- umn and temperature programmed as above. The MS operated at 70 eV ionization energy. Quantitative data were obtained from the electronic integration of the Flame Ioniza- tion Detector (FID) peak areas. Determination of oil composition Identification of the oil components were assigned based on retention indices which were calculated by using retention times of n-alkanes that were injected after the oil at the same chromatographic conditions. The compounds were identified by comparison of their relative retention indices and with those in the literature. In addition, computer searching followed by matching of the mass spectra data with those stored in the com- puter library. The percentage of each com- ponent is presented in Table 1. Mosquito culture Fourth instar larvae Anopheles stephensi was used in this study. All larvae of An. ste- phensi were obtained from laboratory culture of Department of Medical Entomology, Teh- ran University Medical Sciences (TUMS). The Anopheles colony was maintained at 27 °C with 12: 12 light and dark photoperiod in 60±10% relative humidity. Larvicidal bioassay Tests of mosquito larval activity were con- ducted with reference to the standard method recommended by the World Health Organi- zation (WHO 2005). Since the essential oils do not dissolve in water ethanol 99.0% was used as co-solvent. Different concentrations of the essential oils in distillated water and the co-solvent were prepared. The oil-etha- nol-water solution was gently stirred for 30 seconds with a glass rod to ensure a homo- geneous test solution and each glass beaker was left at room temperature. After 15 minutes 20 larvae were taken with a fine mesh strainer and transferred gently to a 400 ml glass beaker. Control group included batches of mosquitoes from the colony exposed to water and the solvent alone. The larvae were exposed to the concentrations of 10, 20, 40, 80 and 160 ppm of essential oil in distilled water for 24 hours at room temperature. In the control beakers only 1 ml of solvent was added to each beaker. Mortality was record- ed after 24 h of exposure while during the test no food was given to the larvae. Each treatment was done with five replicates. Statistical analysis Toxicity and activity were reported as LC 50 and LC90, representing the concentra- tions in ppm that killed 50% and 90%, re- spectively of larvae in 24 h. The LC50 and LC90 values and their 95% confidence in- tervals of each essential oil were calculated by log concentration-probit equation (Finney 1971) using the SPSS 16.0 probit procedure. Controls with mortality between 5–20% were corrected using Abbott’s formula (Abbott 1925). When mortality in controls exceeded 20%, test results were rejected or repeated. Comparison of the LC50 and LC90 values were analyzed using ANOVA Test with SPSS ver- J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 580 http://jad.tums.ac.ir Published Online: October 04, 2016 sion 17.0. Differences between means were considered significant at P≤ 0.05. Result Yields and chemical constituents of essential oil The yields of peel essential oils of C. au- rantium and C. paradisi were 0.7±0.12% and 0.85±0.14% (w/w) based on fresh weight respectively. The chemical composition of C. aurantium peel essential oil is presented in Table 2. A total of 21 compounds were identified representing about 98.62% of the oil. The results revealed that terpenoids in the oil were predominant. The main constit- uents in the C. aurantium peel essential oil were Dl-limonene (94.81%), β-myrcene (1%) and α-pinene (0.65%) respectively. Larvicidal activity of essential oils The LC50 and LC90 values of C. auranti- um and C. paradisi oils against An. stephensi larvae were 31.20 ppm and 73.83 ppm, 35.71 ppm and 70.23 respectively (Table 1). Both peels essential oils at the 80 ppm con- centrations killed more than 90% of the fourth instars larvae (Fig. 1). The mortality rates in the control groups were lower than 5% in all concentrations, no correction were applied. The probit regression lines of An. stephensi exposed to different interval con- centrations of a C. aurantium and C. paradi- si extractions are shown in Fig 2. The statis- tical test (ANOVA) showed that there was no significant statistical difference in mortal- ity rate among two essential oils (P≤ 0.628 ). Table 1. Probit regression line parameters of Anopheles stephensi to peel essential oil extraction of Citrus au- rantium and C. aurantium at different interval concentrations Species A B±SE LC50 , 95% C.I. LC90, 95% C.I. X2 (df) Heterogeneity P-value C. aurantium -5.12 3.43±0.28 28.17 31.20 34.62 63.30 73.83 90.08 3.15 (2) > 0.05 C. paradisi -5.51 3.55±0.29 32.28 35.71 39.62 82.03 70.23 100.47 2.54 (2) > 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, X2= heterogeneity about the regression line. Fig. 1. Percentage of larval mortality of Anopheles. stephensi after treatment with Citrus aurantium and C. paradise J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 581 http://jad.tums.ac.ir Published Online: October 04, 2016 Fig. 2. Probit regression line of Anopheles stephensi exposed to different interval concentrations of a Citrus au- rantium in comparison with C. paradisi essential oils Table 2. Chemical constituents of peel essential oil from Citrus aurantium Concentration (%)KI aCompoundn 0.30909α-Pinene1 0.65947β-Pinene2 1.00958β-Myrcene3 94.81997Dl-Limonene4 0.191016Trans-Ocimene5 0.011029Gamma-Terpinene6 0.041039Linalool Oxide7 0.1310431-Octanol8 0.021044Trans-Linalool Oxide9 0.011045Isoterpinolene10 0.021054Nonanal11 0.441057α-Terpinolene12 0.321234Linalyl acetate13 0.031235Sabinene Hydrate Acetate14 0.031338Neryl Acetate15 0.131358geranyl acetate16 0.031386trans-Caryophyllene17 0.081453Germacrene-D18 0.061536Nerolidol19 0.061929Palmitic Acid20 0.261930Trans-Oleic Acid21 98.62Total Concentration (ppm) J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 582 http://jad.tums.ac.ir Published Online: October 04, 2016 Discussion During past five decades using of chemi- cal insecticides against vector mosquitoes have been developed the resistance in vec- tors to the insecticides and also hazards to the environment (Mittal 1991, Gunasekaran et al. 2004, Vatandoost et al. 2012). Although chemical larval control is considered as a ma- jor component in malaria prevention strate- gies, this part of control has side-effects on human and animal health and also the envi- ronment (Sedaghat et al. 2010, Vatandoost et al. 2012). In order to reduce the dependency on chemical insecticides, alternative methods including botanical insecticides for the con- trol of vectors are considered. Certain plant’s essential oils or extracts have been found ef- fective for mosquito larval control. It is im- portant to identify chemical constituents of the indigenous plans and their efficacies of essential oils as natural larvicides. The yield of oil obtained of C. aurantium was 0.7%. It was nearly as same as reported from Pakistan (Siddique et al. 2011) but less than as reported in Egypt (Hifnaway et al. 2004). Gas chromatography–mass spectrom- etry of peel essential oil of C. aurantium re- vealed the presence of 21 components. In this study, major constituent of peel essential oil of C. aurantium was Dl-limonene, 94.81 % of the oil. In previous studies, various constituents of the oil of C. aurantium were reported. Although limonene is the most abun- dant constituent in the oils obtained from all similar studies, its percentage were varied, based on the origins of the plant (Caccioni et al. 1998, Boussaada and Chemli 2007, Moraes et al. 2009, Hosni et al. 2010). The result is in agreement with the results obtained from other study in Iran (Hosni et al. 2010). The other constituents of the peel oil were α- pinene (0.30%) and β-pinene (0.65%), while the results obtain from a study in Pakistan α- pinene and β-pinene were reported 0.476% and 0.176% respectively (Siddique et al. 2011). The result of the larval bioassay tests showed that essential oils of C. aurantium and C. paradise have a same level of bioac- tivity against An. stephensi larvae. Previous studies have demonstrated the same level of larvicidal activity against mosquito larvae. The LC50 of the essential oil of C. paradise were 47.3 ppm and 85.1 ppm for Ae. aegypti and Ae. albopictus, respectively (Morales- Saldañaet al. 2007). In another study report- ed by Boussaada and Chemli, the content of limonene in Tunisian C. aurantium from 87 % to 92.2% on fresh weight basis (Bous- saada and Chemli 2007). The results nearly similar from the study of Caccioni et al. (1998) which reported limonene (94.3%) myr- cene (1.88%) as the main constituents of C.s aurantium. In another study on the peel oil composition of Brazilian C. aurantium, lim- onene (97.5–98%), myrcene (1.2–1.45%) and octanol (0.34–0.54%) were found as the main constituents (Moraes et al. 2009). The results of constituents of the peel oil of C. auranti- um obtained from this study are similar with all prior studies. Our previous studies on the same strain of An. stephensi larvae revealed that the effica- cy of Eucalyptus camaldulensis, Cupressus arizonica, Coriandrum sativum and Hera- cleum persicum oils were less than the effi- cacy of C. aurantium and C. paradise oils, while the efficacy of Foeniculum vulgare and Kelussia odoratissima are more than the two Citrus (Sedaghat et al. 2010, 2011ab, Vatandoost et al. 2012). According to the classification of Vatan- doost et al. (2012) the level of larvicidal ac- tivity of C. aurantium and C. paradise demon- strated them as active plants. If we accept Cheng et al. (2003) suggestion, these plants should be considered as very active. Based on both above classifications these two plants J Arthropod-Borne Dis, December 2016, 10(4): 577–585 A Sanei-Dehkordi et al.: Chemical Compositions … 583 http://jad.tums.ac.ir Published Online: October 04, 2016 need more attention as they lied in active or very active categories. Results on the larval mortality of C. au- rantium and C. paradise oils against An. ste- phensi confirm their potential to control of the mosquito populations. It seems that the presence of a high amount of Dl-limonene in C. aurantium oil, could demonstrate its effi- cacy against An. stephensi larvae. In brief, this study clearly illustrated the potential use of C. aurantium and C. paradise as natural mosquito larvicides. Conclusion Essential oils from aromatic plants are the complex mixture of constituents with several usages in health and medical sciences. The essential oils of C. aurantium and C. para- dise oils show larvicideal activity against An. stephensi the main vector of malaria in Indo- Persian areas including southern areas of Iran. The results obtained from this study suggest that the limonene of peel essential oil of C. aurantium is promising as larvicide and could be useful in the search for new natural larvicidal compounds. 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