Preparation of a Nanoemulsion of Essential Oil of Acroptilon repens Plant and Evaluation of its Larvicidal Activity Agianst Malaria Vector, Anopheles stephensi http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Original Article Preparation of a Nanoemulsion of Essential Oil of Acroptilon repens Plant and Evaluation of Its Larvicidal Activity agianst Malaria Vector, Anopheles stephensi Samira Firooziyan1,2, Mahmoud Osanloo3, Seyed Hassan Moosa-Kazemi1, Hamid Reza Basseri1, Habib Mohammadzadeh Hajipirloo4, Ali Sadaghianifar2, *Amir Amani 5,6, *Mohammad Mehdi Sedaghat1 1 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Urmia Health Center, Disease Control Unit, Urmia University of Medical Sciences, Urmia, Iran 3 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Fasa University of Medical Sciences, Fasa, Iran 4 Medical Parasitology Department, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran 5 Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran 6 Medical Biomaterials Research Center (MBRC), Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Dr. Mohammad Mehdi Sedaghat, E-mail: sedaghmm@tums.ac.ir; Dr. Amir Amani, E-mail: amani76@gmail.com (Received 07 June 2021; accepted 20 Sept 2021) Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences.Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/ by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited.by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. Abstract Background: Extensive use of chemical larvicides to control larvae, has led to resistance in vectors. More efforts have been conducted the use of natural products such as plant essential oils and their new formulations against disease vectors. Nanoformulation techniques are expected to reduce volatility and increase larvicidal efficacy of essential oils. In this study for the first time, a larvicide nanoemulsion from the essential oil of Acroptilon repens was developed and evaluated against Anopheles stephensi larvae under laboratory conditions. Methods: Fresh samples of A. repens plant were collected from Urmia, West Azarbaijan Prov- ince, Iran. A clevenger type apparatus was used for extracting oil. Components of A. repens essential oil (AEO) were identified by gas chromatography–mass spectrometry (GC–MS). All larvicidal bioassay tests were performed according to the method recommended by the World Health Organization under laboratory condition. Particle size and the morphologies of all pre- pared nanoformulations determined by DLS and TEM analysis. Results: A total of 111 compounds were identified in plant. The LC50 and LC90 values of AEO calculated as 7 ppm and 35 ppm respectively. AEO was able to kill 100% of the larvae in 4 days. Conclusion: The nanoemulsion of AEO showed a weak effect on the larvar mortality. It may therefore be suggested that this kind of nanoemulsion is not appropriate for the formulation as a larvicide. It is important to screen native plant natural products, search for new materials and prepare new formulations to develop alternative interventions with a long-lasting impact. Key words: Acroptilon repens; Nanoemulsion; Larvicidal effect; Vector control; Anopheles stephensi 334 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Introduction Vector borne diseases are infections which are transmitted by the bite of infected arthropod species and account for 17% of all infectious diseases. Every year more than one billion people are infected and more than one million people die from vector- borne diseases including malaria, dengue, schistosomiasis, leishmaniasis, chagas disease, yellow fever, lymphatic filariasis and onchocerciasis (1). The most important diseases transmitted by mosquitoes are malaria, dengue fever, lymphatic filariasis, yellow fever, chikungunya, Zika virus, as well as viral encephalitis (2). Anopheles mosquitoes are widely distributed and found in the tropics temperate regions (3). The most important disease transmitted by Anopheles mosquitoes is human malaria, which is the most important parasitic disease in the world (4). The disease is still a major health problem worldwide, including Iran. Most malaria cases were reported from the south and southeastern of Iran. Plasmodium vivax was the dominant species (5). Anopheles stephensi is an important malaria vector in the Middle East and south Asia (6, 7). To control the disease, larval control is currently being performed in 55 countries (8, 9). The use of natural products is an interesting approach in this regards. Today, there are loads of studies and recommendations on plant extracts and essential oils as larvicides, insecticides and repellents (10, 11). Extracts and essential oils (EOs) are biocompatible and have minimum toxic effects on non-target organisms. Along with many novel formulations, nanoemulsions of pesticides have been considered recently due to their greater efficiency, lesser adverse effects on non-target organisms (12, 13). However, extracts and EOs have volatile components that restrict their use in natural environments (14-16). This can be overcome by formulating them in the form of nanoemulsions. There has been a lot of research recently on EOs as natural larvicides, but there are a few available articles on nanoemulsions as larvicides. In a study, the larvicidal activity of eucalyptus essential oil and its nanoemulsion against Culex quinquefasciatus was investigated. The result showed that the bioactivity of the nanoemulsion was improved than the bulk EO (17). In a study, nanoemulsion of Artemisia dracunculus essential oil showed better larvicidal efficiency on An. stephensi larvae in comparison with its essential oil (18). Likewise, encapsulation of A. dracunculus essential oil in chitosan nanoparticles presented very good larvicidal activity with 9 days residual effect (19). Volpato et al (2016) investigated the effect of essential oil and its nanoemulsion against Alphitobius diaperinus. The nanoemulsion showed a three-fold better effect as compared to the essential oil (20). Balasubramani et al (2017) in their experiments obtained similar results with the nanoformulation of Vitex negundo essential oil compared to its essential oil against Aedes aegypti (21). In a recent study, larvicidal activity of Cinnamomum zeylanicum essential oil was compared with its nanoemulsion. The formulated nanoemulsion showed 32% better larvicidal effect as compared to the essential oil, the residual effect of the formulation was 3 days. These results indicated an increase in larvicidal activity and residual effects of an essential oil nanoemulsion compared to bulk essential oil (11). As the extract of A. repens had very good larvicidal activity against Anopheles stephensi, Culex pipiens and Culex quinquefaciatus in the previous work (22), we decided to extract its essential oil and provide the nanoformulation in order to investigate their larvicidal effect against An. stephensi larvae. Materials and Methods Collection, identification and extraction of Acroptilon repens Fresh samples of A. repens were collected in Jun- Jul 2018 from Urmia, West Azarbaijan province, Iran (45.08º E, 37.55º N, elevation ~ 1332 m above sea level) (Fig.1). 335 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Collection and identification of plant Acroptilon repens plants were collected (Fig. 2), rapidly transferred to the laboratory and then was identified by experts in Department of Plant Sciences, Tehran University. Extraction of essential oil All collected plants were washed with water, then shad dried. Dried samples were hydrodistilled, using clevenger type apparatus for five hours. The extracted oil dried over anhydrous sodium sulfate. In total, 65 ml extracted oil obtained from 650 kg of dried plant. To prevent degradation and oxidation, the essential oil was stored in dark glass containers, completely away from sun light at 4-8 °C. Analysis of essential oil by gas chromatog- raphy–mass spectrometry (GC-MS) GC-MS analysis used to identify compounds of the essential oil. The essential oil diluted using hexane with the specifications given in Table 1. The compounds of the essential oil were analyzed using GC-MS and compared with standard mass spectra available in the device library. Mosquito rearing Anopheles stephensi larvae were reared in the insectary at 29 ± 1˚C with relative humidity of 70 ± 5% under 12 h light/12 h dark conditions. The cages for keeping mosquitoes were wooden cubes with dimensions of 30 cm × 30 cm × 30 cm, covered with fine mesh. The stock culture of adult An. stephensi fed twice a week on sheep blood (artificial feeding). The egg rafts laid transferred to enamel larval trays. The larvae were fed with fish food. Preparation of nanoemulsion In this study, surfactant (Tween 80) and co-surfactant (Span 20) were stirred for 6 minutes at 600 rpm. The essential oil was then added at 90% lethal concentration of the bulk essential oil and stirred for 10 minutes at 600 rpm. Water was then added dropwise and stirring was continued at 600 rpm for 38 minutes. Ten different nanoemulsion preparations having a constant amount of essential oil (1.4 %) and different amounts of surfactant (2 to 9.2 %) and co-surfactant (0.8 %). The nanoemulsion stored in a dark place at room temperature for 24 hours, then checked visually for signs of phase Fig. 1. Collection site of plant Acroptilon repens in Urmia, West Azarbaijan Province, Iran Fig. 1. Collection site of plant Acroptilon repens in Urmia, West Azarbaijan Province, Iran 336 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... separation, precipitation or creaming. Dynamic light scattering (DLS, K-ONE. LTD, Korea) used to determine the particle size (PS) of the prepared nanoformulations. Transmission electron microscopy (TEM) used to confirm the PS and to investigate the morphology of the particles. Determining the larvicidal efficacy Larvicidal bioassays performed according to the WHO guideline. Logarithmic dilutions prepared from bulk essential oil by dissolving in ethanol. All the nanoemulsion/ bulk samples diluted 200 times before performing the larvicidal tests (23). Third and early 4th instars larvae were used. One ml of the essential oil was added to 249 ml of chlorine-free (pH=7) water and stirred and 25 healthy larvae added to the containers. Containers were covered and after 24 hours, the number of living and dead larvae counted. Fig. 2. Acroptilon repens Fig. 2. Acroptilon repens Table 1. Analysis conditions and specifications of GC-MS device Instrument Specifications Manufacturer company Agilent Technologies 1. GC system 7890A 2. Mass Selective Detector 5975C VL MSD with Triple-Axis Detector 3. Ion source Electron Impact (EI) 70eV 4. Analyzer Quadrupole 5. Column Rtx 5 MS -Length 30m -I.D. 0.250 mm -Film thickness 25 μm Conditions 1. Injection port temperature 230˚C 2. Ion source temperature 230˚C 3. Carrier gas He 99.999% 4. Sample volume 0.2 μL Temperature Program Initial temperature (ºC) 40 Initial time (min) 1 Program rate (ºC/min) 3 Final temperature (ºC) 270 Final time (min) 10 Split ratio (ml/min) 100 Septum purge (ml/min) --- Flow rate (ml/min) 1 Table 1. Analysis conditions and specifications of GC-MS device 337 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Determining the duration of action of bulk and nanoemulsions of A. repens essential oil To determine the duration of larvicidal activity, according to the instructions of the WHO, the formulation prepared for the larvicidal test diluted 100 times (23). One ml of specified concentrations of bulk or nanoemulsion samples added to 249 ml of chlorine-free (pH=7) water, then, 25 live larvae were added to the solution. After 24 hours, the number of dead/ live larvae was counted, without changing the solution; the larvae (dead and live) were removed from the containers, followed by the addition of 25 new live larvae in the containers. The larvae were replaced for 8 days. All the larvicidal bioassays repeated 16 times in four different replicates. In each replicate, two control groups containing ethanol were considered. Statistical analysis The lethal concentrations of 50% and 90% (LC50 and LC90) were calculated using Probit analysis (24). The regression line plotted using Excel 2007 software. If mortality of the control group was less than 5%, the data from the bioassay tests were considered correct. When the control mortality was between 5- 20%, it was corrected using the Abbott formula (24). If the larvae became pupae or the larvae mortality were more than 20% in the control group, the test was repeated. Results Determination of chemical composition of A. repens essential oil Components of AEO identified by GC– MS analysis. One hundred and eleven components were determined, with five major components including Caryophyllene oxide (12.055%), α-Cubebene (12.054%), 1-Heptadecene (5.181%), delta-Cadinene (3.771%) and β-Cubebene (3.771%) as listed in supplementary data (Table 2). Characterization of AEO nanoemulsion From preliminary studies to find on the optimum nanoemulsion (i.e., highest stability and lowest particle size), a nanoemulsion preparation with 6.8% Tween 80, 0.8% Span 20, 1.4% AEO and water was prepared. Figure 3 shows DLS results of the nanoemulsion with d50= 106 nm. The morphology of nanoemulsion parti- cles was determined using transition electron microscopy (TEM) (Fig. 4). The results show that the nanoemulsion was well-formed and the particles were almost spherical. The AEO nanoemulsion did not show any sign of phase separation after 4-month storage (4 °C and room temperature) and centrifugation (25000 rpm, 30 min). Larvicidal bioassay of A. repens essential oil The results of larvicide activity of six different concentrations of AEO are shown in Figure 5. Mortality rate at 3.125 ppm was 0% and increased to 100% at 50 ppm. There was no mortality in the control groups. In regression line, a positive correlation was observed between essential oil concentrations and the probit mortality (Fig. 6). The LC50 and LC90 values of AEO against An. stephensi larvae calculated as 7 and 34 ppm, respectively. Figure 7 shows comparison of the residual larvicidal properties. AEO killed 100% of the larvae in the first four days of the experiment. After the 4th day, larval mortality decreased and reached 76%. AEO nanoemulsion showed weaker activity. It had 84% mortality on the first day and the mortality rate decreased to 0% on the 7th day. To compare larvicidal activity of AEO with AEO nanoemulsion against An. stephensi larvae, equal concentrations of AEO used in the short time test (24 hours). The larvicidal effects of AEO were 88%, while the nanoemulsion properties of AEO reduced to 44% (Fig. 8). Figure 9 shows the particle size of the nanomeulsion after 200 times dilution, showing instability for the preparation after dilution. 338 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Table 2. Chemical composition of the essential oil of Acroptilon repens No. RT (min) Compound Peak area % Quality Mol Weight (amu) 1 37.993 Caryophyllene oxide 503332611 12.055 96 220.183 2 29.563 α-Cubebene 503308397 12.054 99 204.188 3 41.823 1-Heptadecene 216337204 5.181 99 238.266 4 35.537 delta-Cadinene 188186597 4.507 98 204.188 5 30.091 β-Cubebene 157435883 3.771 97 204.188 6 31.287 Caryophyllene 155313108 3.720 99 204.188 7 28.316 α-Cubebene 148603368 3.559 98 204.188 8 38.858 3-Undecyne 128983727 3.089 55 152.157 9 47.078 2-Pentadecanone 108810045 2.606 96 268.277 10 33.756 Tricyclo[2210(2,6)]heptane-3,5-diol 96224253 2.305 91 204.188 11 40.881 1,3-Cyclooctadiene 93566390 2.241 74 108.094 12 34.201 1-Pentadecene 91513407 2.192 99 210.235 13 38.285 Dihydrotanshinone I 87244445 2.090 91 220.183 14 37.172 [1,5]Naphthyridine-4-carbaldehyde 77809615 1.864 58 220.183 15 38.673 2(1H)-Naphthalenone 64550157 1.546 86 220.183 16 41.231 Azulene 63504092 1.521 97 198.141 17 38.063 β-Copaen 62409995 1.495 70 220.183 18 30.689 4,4-Dimethyl-3 56695349 1.358 47 202.172 19 33.406 delta-Elemene 53675197 1.286 58 204.188 20 41.422 Methyl α-oxo-7-azaindole-3-acetate 50884172 1.219 59 218.167 21 32.655 5,9-Undecadien-2-one 46366238 1.110 90 194.167 22 40.652 Bicyclo 45358016 1.086 89 204.188 23 29.881 β-Damascenone 45288877 1.085 98 190.136 24 36.249 Calacoene 44746403 1.072 78 172.125 25 25.752 1-Tridecene 41473191 0.993 98 182.203 26 36.612 Caryophyllene oxide 39040647 0.935 60 220.183 27 33.972 β-Selinene 37808861 0.906 99 204.188 28 39.526 Naphthalene 36960428 0.885 90 204.188 29 30.212 3,5-Octadiene 33754464 0.808 64 194.203 30 39.869 10,10-Dimethyl-2,6-dimethylenebicyclo 33082573 0.792 99 220.183 31 33.546 β-Selinene 32759075 0.785 96 204.188 32 40.048 Bicyclo 30063246 0.720 84 204.188 33 34.354 delta-Cadinene 29461311 0.706 80 204.188 34 32.922 Bicyclo[221]heptane 27543459 0.660 98 204.188 35 60.641 Tricosane (CAS) 25909857 0.621 98 324.376 36 35.212 delta-Cadinene 25336335 0.607 62 204.188 37 36.803 Bicyclo[310]hexane 24755522 0.593 42 136.125 38 55.424 Phytol 23404833 0.561 90 296.308 39 37.579 Cyclohexane 22932610 0.549 84 192.188 40 75.287 Nonacosane 21848026 0.523 99 408.47 41 51.238 Hexadecanoic acid 20517153 0.491 99 256.24 42 34.557 Naphthalene 19954822 0.478 99 204.188 43 32.77 Trimethylcyclohex 19947825 0.478 80 278.134 44 70.75 Heptacosane 18873599 0.452 95 380.438 45 67.226 Benzenedicarboxylic acid 18814084 0.451 91 211.012 46 30.925 Methanoazulene 18182957 0.435 99 204.188 47 40.474 Isoaromadendrene epoxide 18089104 0.433 43 220.183 48 31.904 Bicyclo[311]hept 17605938 0.422 98 204.188 Table 2. Chemical composition of the essential oil of Acroptilon repens 339 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Continued Table 2. Chemical composition of the essential oil of Acroptilon repens No. RT (min) Compound Peak area % Quality Mol Weight (amu) 49 30.409 7-Methanoazulene 15474913 0.371 99 204.188 50 35.823 Naphthalene 14843167 0.356 99 204.188 51 32.515 Khusimene 14686454 0.352 91 204.188 52 21.929 Decanal 13680181 0.328 91 156.151 53 33.851 Tricyclo 13035063 0.312 91 204.188 54 39.717 Dimethyl-2 12491034 0.299 83 220.183 55 40.347 4-Methanoazulene 12398829 0.297 55 204.188 56 34.977 Tridecanal 12181378 0.292 94 198.198 57 34.474 Pentadecane 11697698 0.280 93 212.25 58 49.457 13-Pentadecatrien-2-one 11566338 0.277 86 262.23 59 25.161 Vitispirane 11436343 0.274 98 192.151 60 28.914 Cycloisosativene 11154885 0.267 97 204.188 61 40.226 gamma-Selinene 11037619 0.264 64 204.188 62 41.097 Quinoline, 2,6-dimethyl- 10239966 0.245 35 157.089 63 65.877 Heneicosane 9939147 0.238 91 296.344 64 41.988 Heptadecane 9896521 0.237 97 240.282 65 36.491 2-Methyl-6-nitrophenol 9850609 0.236 53 153.043 66 44.463 Alloaromadendrene oxide-(2) 8884878 0.213 83 220.183 67 32.146 4-Dimethylaminopyridin-2-amine 8576066 0.205 52 137.095 68 39.316 6-Methoxy-1-acetonaphthone 8242200 0.197 78 200.084 69 48.751 Nonadecane (CAS) 8087481 0.194 98 268.313 70 26.408 1H-Indene 7844665 0.188 92 174.141 71 30.835 1H-Cycloprop[e]azulene 7747134 0.186 99 204.188 72 54.941 Heneicosane 7588497 0.182 99 296.344 73 37.045 Naphthalene 7339014 0.176 80 172.125 74 42.192 Vulgarol B 7325012 0.175 55 220.183 75 44.164 Ledene oxide-(II) 7233022 0.173 60 220.183 76 39.138 Trimethyl-2'-methylidene-9'-oxabicyclo 7090758 0.170 41 220.146 77 43.025 2-Dodecen-1-yl(-)succinic anhydride 7006122 0.168 30 266.188 78 45.735 Eicosane 6974224 0.167 38 282.329 79 28.443 Naphthalene 12 dihydro 1 1 6 trimethyl 6527008 0.156 97 172.125 80 39.221 Tricyclo 6429488 0.154 38 220.183 81 45.43 Octadecane 6266398 0.150 98 254.297 82 31.618 Germacrene-D 5570492 0.133 98 204.188 83 43.407 Valerenol 5558781 0.133 70 220.183 84 43.559 Isopropylidene 5353049 0.128 42 218.167 85 49.673 Hexadecanoic acid 5318288 0.127 98 270.256 86 22.323 Pentylthiophene 5122791 0.123 83 154.082 87 54.547 1-Heptadecanol 5095930 0.122 95 256.277 88 46.066 Bicyclo[1310]hexadecan-2-one 4816216 0.115 55 236.214 89 13.595 dl-Limonene 4755721 0.114 99 136.125 90 36.129 α-Calacorene 4672444 0.112 38 200.157 91 51.906 Eicosane 4631346 0.111 96 282.329 92 28.997 Cycloisosativene 4487495 0.107 99 204.188 93 42.554 Tetradecanal 4420395 0.106 91 212.214 94 48.178 Cyclotetradecane 4361575 0.104 90 196.219 95 16.483 Benzene 4265136 0.102 96 132.094 96 22.075 Naphthalene, 1,2,3,4-tetrahydro 4132000 0.099 97 174.141 97 44.666 7,8-Dihydropyran 4116843 0.099 50 173.084 98 27.68 Benzene, 1,2,3,4-tetramethyl- 4066183 0.097 46 134.11 99 11.991 Furan, 2-pentyl- 3750856 0.090 91 138.104 340 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Continued Table 2. Chemical composition of the essential oil of Acroptilon repens No. RT (min) Compound Peak area % Quality Mol Weight (amu) 100 32.019 Aromadendrene 3697814 0.089 99 204.188 101 33.189 Widdrene 3626118 0.087 83 204.188 102 13.423 Benzene, 1-methyl-4-(1-methylethyl)- 3546573 0.085 97 134.11 103 57.836 Docosane 3527160 0.084 94 310.36 104 17.214 Nonanal 3429151 0.082 91 142.136 105 56.277 4,4,6-Trimethyl 3326248 0.080 43 140.12 106 42.923 4,4-Dimethyl-3 3080628 0.074 89 202.172 107 63.294 Tetracosane 2968794 0.071 97 338.391 108 32.324 Bicyclo[311]heptane 2911317 0.070 60 204.188 109 43.311 4-Tetradecene 2856951 0.068 84 196.219 110 23.316 cis-3-Hexenyl isovalerate 2576062 0.062 72 184.146 111 43.19 2-Cyclopenten-1-one 2205731 0.053 90 164.12 Fig. 3. DLS results of AEO nanoemulsion Fig. 3. DLS results of AEO nanoemulsion Fig. 4. Transition electron microscopy (TEM) image of AEO nanoemulsion Fig. 4. Transition electron microscopy (TEM) image of AEO nanoemulsion 341 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Fig. 5. Larvicidal activity of AEO against Anopheles stephensi 0 10 20 30 40 50 60 70 80 90 100 3.125 6.25 12.5 25 50 0 48 80 84 100 M or ta lit y% Concentration(ppm) Fig. 5. Larvicidal activity of AEO against Anopheles stephensi Fig. 6. Probit regression line of AEO against Anopheles stephensi larvae y = 34.048ln(x) - 23.595 0 20 40 60 80 100 120 1 10 100 L ar va l M or ta lit y (p ro bi t) Concentration (log) 50 nm Fig. 6. Probit regression line of AEO against Anopheles stephensi larvae Larvicidal effects of AEO and AEO nanoemulsion Fig. 7. Comparison of residual larvicidal effect of AEO vs. AEO nanoemulsion (after diluting 100 times) during an 8-day study 0 20 40 60 80 100 1 2 3 4 5 6 7 8 100 100 100 100 92 84 82 76 84 76 68 56 40 16 0 0 M or ta lit y % Days AEO AEO nanoemulsion Fig. 7. Comparison of residual larvicidal effect of AEO vs. AEO nanoemulsion (after diluting 100 times) during an 8-day study 342 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... Fig. 8. Comparison of larvicidal activity of AEO vs. AEO nanoemulsion (after diluting 200 times) during an 24 88 44 0 10 20 30 40 50 60 70 80 90 100 AEO AEO nanoemulsion M or ta lit y % Fig. 8. Comparison of larvicidal activity of AEO vs. AEO nanoemulsion (after diluting 200 times) during an 24 Fig. 9. DLS results of AEO nanoemulsion after dilution Fig. 9. DLS results of AEO nanoemulsion after dilution Discussion Today, associated with extensive use of various chemical pesticides, serious damages have been observed in the environment and non-target organisms which is being carefully considered by international organizations such as United States Environmental Protection Agency (USEPA), World Health Organization (WHO) and Food and Agricultue Organization (FAO) (25). In addition, frequent use of insecticides has led to their resistance for vectors (26). To reduce environmental damages and increase the effectiveness of insecticides on target organisms, the use of novel preparations such as nano-formulations has been suggested (10). In this study the most components of AEO were identified in comparison with the similar studies. Total number of components in AEO in earlier studies varied from 11 to 77 compounds (27-32) while we were able to identify 111 components in the essential oil due to timely GC analysis. Our research showed LC50 and LC90 of AEO as 7 and 34 ppm against An. stephensi, respectively. Reviewing other reports have shown different values for other essential oils against An. stephensi. Depending on the obtained results, LC50 values are summarized as follows: LC50 < 10 ppm: 1 EO (Kelussia odoratissima), 10 ppm < LC50 < 50 ppm: 22 EOs (C. zeylanicum (11), Ar. dracunculus, Platycladus orientalis, Tagetes patula, Ferulago carduchorum, Chloroxylon 343 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... swietenia, Ipomoea cairica, Feronia limonia, Chloroxylon swietenia, Foeniculum vulgare, Satureja bachtiarica, Bunium persicum, Plectranthus amboinicus, Citrus aurantium, Plectranthus mollis, Achillea kellalensis, Citrus paradisi, Anethum graveolens, Achillea wilhelmsii, Zingiber nimmonii, Zingiber cernuum and Blumea eriantha) 50 ppm < LC50 < 100 ppm: 14 EOs (Murraya exotica, Syzigium aromaticum, Zanthoxylum armatum, Zhumeria majdae, Origanum scabrum, Boswellia ovalifoliolata, Lavandula gibsoni, Origanum vulgare, Lawsonia inermis, Cionura erecta, Cupressus arizonica, Trachyspermum ammi, Eucalyptus camaldulensis, Coccinia indica) and LC50 > 100 ppm: 9 EOs (Kadsura heteroclita, Stachys byzantina, Heracleum persicum, Coriandrum sativum, Stachys setifera, Thymus vulgaris, Stachys inflata, Ajuga chamaecistus tomentella and Cedrus deodara) (10). According to proposed categories of larvicidal activity of plant essential oils against mosquito larvae, AEO lies in the third category as an active plant (33). In previous work, LC90 and LC50 of A. repens extract against An. stephensi were 0.37 ppm and 3.39 ppm, against Culex pipiens were 3.5 ppm and 60 ppm and against Cx. quinquefaciatus were 4 ppm and 39.7 ppm, respectively (22). With the help of nano-techniques, the stability of essential oils in nature increases. Additionally, nanoproducts cause faster absorption in the target insect (25, 34). In a report, nanoemulsions of Azadirachta indica essential oil with different particle sizes (31, 93 and 251 nm) were prepared and tested against Cx. quinquefasciatus. The nanoemulsion with smallest particle size was found to be the most effective larvicidal agent (35). In another study, nanoemulsion of Ar. dracunculus essential oil was investigated against An. stephensi. The size of the prepared nanoemulsions was 12 to 291 nm. Similar to the above, larvicidal properties of the nanoemulsion increased significantly with decreasing droplet size (18). Previous studies had shown good results of new nanoformulations as larvicides (11), although all prepared nanoformulations had no similar effects. It is possible that different effects can be observed among different plant natural products and their formulations. In this experiment, the comparison has been made between the larvicidal activity of bulk essential oil and its nanoemulsion against one of the main vectors spreading malaria, An. stephensi. In this study, AEO showed complete mortality of larvae for up to 4 days, while its corresponding nanoemulsion failed to indicate 100% mortality even on the first day after diluting 100 times. Besides, the bulk preparation showed more larvicidal effect compared with the nanoemulsion after diluting 200 times (i.e. 88% vs. 44%). In a similar study, nanoemulsion of Artemisia dracunculus essential was broken or at least showed substantial changes in its nanostructures; it was not able to show a change in larvicidal activity of the essential oil (18). In another study, after dilution, by breaking nanostructure of Anethum graveolens essential oil, practically, no difference may be determined between nanoemulsion and bulk essential oil (36). In total, our nanoemulsion preparation failed to show good efficacy compared with the bulk essential oil. To investigate the possible reason, we measured the particle size after 200 dilutions and found that the nanoemulsion breaks up after dilution. In other studies, nanoemulsions of essential oils have been tested against larvae. The results appear to be promising. For instance, nanoemulsion of Copaifera duckei (37), Rosmarinus officinalis (32) and Ocimum basilicum (38) have shown potential against Ae. aegypti however, considering the reports, the nanoemulsions have not been diluted 100 or 200 times (as recommended by WHO). Additionally, in these studies, the results of nanoemulsions have not been compared with the bulk essential oils. It is arguable that by performing the studies similar to ours, the nanoemulsions would not indicate positive results. Based on the result of the current study, difficulty in obtaining AEO and the negative larviciding results, we therefore do 344 http://jad.tums.ac.ir Published Online: Sep 30, 2021 J Arthropod-Borne Dis, Sep 2021, 15(3): 333–346 S Firooziyan et al.: Preparation of... not recommend considering AEO as a good candidate for the next studies. However, it is suggested that for performing the larvicidal studies, nanoemulsions which are stable after 200 dilutions, should be considered. Different extractions of the following Iranian native plants were evaluated against main malaria vector, An. stephensi, such as Mentha spicata, Cymbopogon olivieri, Azadirachta indica, Melia azedarach, Tagetes minuta, Calotropis procera, Eucalyptus camaldulensis, Cupressus arizonica, Thymus vulgaris, Lawsonia inermis, Cedrus deodara, Cionura erecta, Bunium persicum, Carum carvi, Artemisia dracunculus, Rosmarinus officinalis. (39-44). World Health Organization recommended several biological and chemical insecticides for mosquito larval control including: Bacillus thuringiensis H-14, B. spahericus, Chlorpyrifos, Chlorp- yrifos-methyl, Deltamethrin, Diflubenzuron, Etofenprox, Fenitrothion, Fenthion, Fuel oil, Malathion, Methoprene, Permethrin, Phoxim, Pirimiphos-methyl, Pyriproxyfen, Temephos, and Triflumuron (45). Monitoring and mapping of insecticide resistance is appr-opriate measure for vector control. 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World Health Organization. (2006). Pesticides and their application for the control of vectors and pests of public health importance, pp125. Preparation of a Nanoemulsion of Essential Oil of Acroptilon repens Plant and Evaluation of its Larv Abstract Key words Introduction Materials and Methods Collection, identification and extraction of Acroptilon repens Collection and identification of plant Extraction of essential oil Analysis of essential oil by gas chromatography-mass spectrometry (GC-MS) Mosquito rearing Preparation of nanoemulsion Determining the larvicidal efficacy Determining the duration of action of bulk and nanoemulsions of A. repens essential oil Statistical analysis Results Determination of chemical composition of A. repens essential oil Characterization of AEO nanoemulsion Larvicidal bioassay of A. repens essential oil Discussion Conclusion Acknowledgment Conflict of Interest References