Introduction

According to a report by WHO, just in 2015, 212 million of new cases of malaria were identified, with 429,000 death caused by the disease around the world (WHO 2016). Anopheles stephensi is major vector of spreading malaria, especially in Eastern Mediterranean and South-East Asia regions of WHO (Maheswaran and Ignacimuthu 2015; WHO 2016).

Continuous of applying chemical larvicides for control of mosquito-borne diseases, such as malaria, has led to occurring resistance against the mosquitoes, especially in An. stephensi. Environment pollution is a second outcome of this constant use (Poopathi et al. 2002; Soltani et al. 2015; Vatandoost and Hanafi-Bojd 2005; Vatandoost et al. 2005). EOs are naturally extracted aroma compounds, with wide applications such as flavoring additives, medicines, antioxidants, antifungals/bacterials, and larvicides. During the past decade, EO-based larvicides are proposed as suitable alternatives for industrial ones (Donsì and Ferrari 2016; Govindarajan et al. 2017; Keyal et al. 2016; Langeveld et al. 2014; Oliveira Fde et al. 2014; Osanloo et al. 2017b; Pavela 2015).

To retain their biological activity, vaporization of volatile components of EOs should be prevented; thus, EOs need to be formulated (Bakkali et al. 2008; Buranasuksombat et al. 2011; Osanloo et al. 2017a). Nanoemulsions are fine oil-in-water dispersions, having droplet in the size of < 200 nm, with increased bioactivity, due to subcellular size and better diffusion (Donsì et al. 2012; Esmaeili et al. 2016; Khani et al. 2016; Mishra et al. 2017). From the literature, nanoemulsions of some EOs such as rosemary, eucalyptus, basil, and copaiba have been prepared as larvicides (da Rodrigues et al. 2014; Duarte et al. 2015; Ghosh et al. 2013; Sugumar et al. 2014). In our previous report, by preparation of nanoemulsion of Tarragon EO with PS of ~15 nm, LA significantly improved at 18 ppm, i.e., 83% < 92%, against An. stephensi (Osanloo et al. 2017a). In another study, nanoemulsions of Neem EO were prepared with different sizes (i.e., 31, 93, and 251 nm) and showed maximum LA when PS was 31 nm, against Culex quinquefasciatus (Anjali et al. 2012).

Anethum graveolens (Umbelliferae family), known as Dill, is a widespread plant which is used in foods and pharmaceuticals as an antibacterial, anti-inflammatory, and antifungal agent (Chen et al. 2014; Kumar et al. 2017; Ma et al. 2015; Orhan et al. 2013; Snuossi et al. 2016). EO of Dill has also shown LA against some larvae specious: LC at 50 and 90% against Aedes aegypti has been reported as 20.2 and 34.7 μg/mL, respectively (Promsiri et al. 2006). In another report, Dill EO at concentration of 0.1 mg/mL had 90% mortality against Aedes albopictus (Seo et al. 2015).

In this study, nanoemulsion was prepared using spontaneous emulsification, which is a mild procedure for preparing nanoemulsions from different oils, including EOs. This method uses optimized amounts of oil, surfactant, and water, and no mechanical force such as homogenizer or ultrasound is employed as they can lead to evaporation of volatile components (Bouchemal et al. 2004; Osanloo et al. 2017a). For the first time, nanoemulsion of Dill EO was prepared and optimized, then, its LA against 3rd and 4th instar larvae of An. stephensi (Beech-Lab strain) was compared with its bulk form.

Materials and methods

Materials

Dill EO was purchased from Barij Essence Pharmaceutical Company, (Iran). Tween 20 and ethanol were obtained from Merck chemicals (Germany). Third and fourth instar larvae of An. stephensi (Beech-Lab strain) were used in this research, obtained from the Department of Medical Entomology, Tehran University of Medical Sciences. This strain has been maintained in the laboratory without exposure to insecticides for 30 years.

Determining ingredients of EO of dill by GC-MS analysis

The GC-MS Analyses were performed using a 6890 GC system coupled with 5973 network mass selective detector (Agilent Technologies, USA). Separation of the EO components was carried out on an HP-5MS silica fused columns (30-m length, 0.25-mm internal diameter, and 0.25-μM film thickness 5% phenyl-methylpolysiloxane). The GC-MS column temperature was programmed as follows: initial temperature was set at 40 °C and fixed for 1 min, then, increased with rate of 3 °C/min to final temperature of 250 °C and hold for 60 min. Temperature of injection port and detector was fixed at 250 and 230 °C, respectively. Other instrument parameters were set as split flow 25 mL/min, septum purge 6 mL/min, and column flow rate 1 mL/min. Helium gas with purity of 99.99% was used as carrier gas. Mass spectra were taken at full scan mode and 70 eV ionization energy with scanned mass range at 50–350 m/z.

Determination of components of EO was performed by comparing their retention indices (RIs) determined with reference to a homologous series of C9–C24 n-alkanes. Firstly, this was confirmed by chromatographic injection of available analytical standard compounds (C9–C24 n-alkanes) and comparison of their retention times with those obtained for the EO. If standard compounds were not available, the identification was carried out by comparison with traditional retention indices. The identification was also confirmed by comparison of their mass spectra with those stored in the Wiley7n.l MS computer library. The linear temperature-programmed retention indices (RIs) of all the constituents were calculated from the gas chromatogram by interpolation between bracketing n-alkanes (Eq. (1).

$$ RI=100\left[\frac{tR(i)- tR(z)}{tR\left(z+1\right)- tR(z)}+z\right] $$
(1)

where z is the number of carbon atoms in the smaller n-alkane, and tR(i), tR(z), and t are the retention times of the desired compound, the smaller n-alkane and the larger n-alkane, respectively. In addition, the search match factor (SMF), rank number (RN) in the mass library, and five highest peaks in the mass spectra were prepared and used for identification of the components.

Evaluation of LA

LA of EO was evaluated according to recommended method by WHO, with some modification, against An. stephensi (WHO 2005). In brief, solutions (1:200) of different concentrations of bulk Dill EO (EO dissolved in ethanol) or nanoformulations were prepared in containers having no chlorine water. Subsequent to homogenizing them with specific rubber probe, batches of 25 larvae of An. stephensi were added to all containers. After 24 h of exposure, counted dead larvae as well as LC at 50 and 90% were calculated using probit analysis and SPSS software (v22). Tests were repeated 12 times in 3 different replicates in recommended conditions, (25–28 °C and 12 h L: 12 h D photoperiod). For increasing accuracy, the tests were discarded if mortality in control groups (ethanol only added) increased from 5%.

Preparation of nanoemulsions

Many components of Dill EO are volatile; thus, spontaneous method (without using mechanical force) was used for preparation of nanoemulsions. Different amounts of tween 20 (5–30%) with/out ethanol (up to 30%) were mixed by Dill EO (fixed at 1.2%, comparable with calculated LC90%) at 600 rpm and room temperature. Then, deionized water was added gradually up to 5 mL and stirred for 15 min, for preparation of nanoemulsions.

Analyzing PS of nanoemulsions

PS and PSD (particle size distribution) of prepared nanoemulsions were determined using DLS (dynamic light scattering, scatteroscope, K-ONE.LTD, Korea) and confirmed by TEM (transition electron microscopy, LEO 906E, Zeiss, Germany). PSD was calculated using Eq. 2.

$$ \mathrm{PSD}=\sqrt{d\left(75\%\right)/d\left(25\%\right)} $$
(2)

d median diameter of particles (percent of cumulative).

For evaluation of physical stability, optimum formulation (F9) was ultra-centrifuged in 25,000 rpm at specific temperatures (i.e., 4 °C and room temperature). In another test, the nanoformulation was stored at mentioned temperatures for 30 days, then, visually checked for any creaming, precipitation, or phase inversion.

Comparison of larvicidal activity of Dill EO vs. selected nanoemulsions

Nanoemulsions with PS < 20 nm and PSD < 2 were selected (i.e., F2 and F9) and their LA was compared with similar concentration of bulk Dill EO (1.2% dissolved in ethanol). By adding 1 mL from each sample to test containers, concentration of oil was eventually fixed at 60 ppm. Two control groups were also considered: nanoformulations without Dill EO (i.e., micelles of tween) and ethanol only. For comparing the LA of nanoemulsions with bulk Dill EO, SPSS software and ANOVA with 95% confidence intervals were used.

Results and discussion

Determining ingredients of Dill by GC-MS analysis

In total, 39 components for the EO were identified by GC-MS analysis (see Table 1). Five ingredients with high amounts were detected: p-Cymenealpha (20.81%), alpha-Phellandrene (20.75%), Carvone (10.97%), Dill ether (9.88%), and cis-Sabinol (3.61%).

Table 1 Components of Dill essential oil, identified by GC-MS analysis
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Evaluation of LA of Dill EO

Results of LA of bulk Dill EO at different concentrations (10–100 ppm) against An. stephensi are demonstrated in Fig. 1. LA appeared from 20 ppm and increased by arising concentration of EO. Calculated LC50 (38.8 ppm) and LC90 (65.0 ppm) and probit equation are given in Table 2.

Fig. 1
figure 1

Evaluation of larvicidal activity of bulk Dill EO against An. stephensi

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Table 2 Results of probit analysis for larvicidal activity of Dill EO against An. stephensi
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In the literature, LA of many EOs against An. stephensi can be found. For instance, LC50 of EOs of Citrus aurantium, Citrus paradise, and Nigella sativa are reported as 31.20, 35.71, and 53.9 ppm, respectively (Raj et al. 2015; Sedaghat et al. 2016).

Repellency activity of Dill EO against different specious of mosquitoes (such as Ae. aegypti, An. stephensi, and Cx. quinquefasciatus) has been evaluated (Amer and Mehlhorn 2006b). LA of 41 herbal EOs, including Dill EO, has been reported against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus, with LC50 of ~100 ppm against An. stephensi (Amer and Mehlhorn 2006a). However, we could not find a comprehensive report showing LC50 and LC90 and concentration with perfect effect of Dill EO against An. stephensi.

Vatandoost et al. (Vatandoost et al. 2012) classified EOs into 5 groups according to larvicidal properties. LA of bulk Dill EO lies in active group of this classification, thus, has merits for further investments, such as preparing its nanoformulation.

Preparation of nanoemulsion of Dill EO

Based on the LA studies of Dill EO, developing a nanoformulation from Dill EO has the potential to improve its efficacy, without observing environmental pollution. Components ratio and size of various prepared nanoemulsions of Dill EO are depicted in Table 3. PS and PSD ranged 10.7–1880 nm and 1.3–8.2, respectively. By increasing concentration of tween (without ethanol) from 5 to 20%, PS increased (i.e., 211–1880 nm, see samples F1, F3, F5, and F7). However, by further increasing the concentration of tween to 30% (i.e., samples F9 and F11), PS decreased. Interestingly, at 30% tween concentration, PSD increased which is probably due to formation of micelles without oil in core of the particles. Above 30% tween concentration, a gel-like preparation was prepared (i.e., very high viscosity), thus, preparing nanoemulsions with higher concentrations of tween was not performed.

Table 3 Components ratio of prepared nanoemulsions of Dill essential oil (EO was fixed at 1.2%, distilled water was added to all the samples up to desired volume of 5 mL)
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Ethanol as co-surfactant helps improved dispersion of tween and oil; thus, by adding ethanol to the formulations, PS suddenly decreased (e.g., PS of F1 (211 nm), decreased to 17.1 nm (i.e., F2)). A balance between ingredients is necessary for obtaining smallest PS and PSD values in nanoemulsions. In previous researches, nanoemulsion with smaller size showed better larvicidal activity (Anjali et al. 2012; Osanloo et al. 2017a). Also, smaller PSD values are often preferred to improve physical stability (Esmaeilzadeh-Gharehdaghi et al. 2014; Sattler 2010), performance (Akbarzadeh et al. 2012; Cui et al. 2009), and loading capacity (Sinko 2006). Thus, in this study, nanoemulsions with smallest PS (i.e., < 20 nm) and PSD (i.e. < 2) were selected for investigation of their larvicidal activity (i.e., F2 and F9).

Comparison of LA of bulk Dill vs. selected nanoemulsions

DLS results of undiluted form of the selected nanoemulsions (i.e., F2 and F9) are illustrated in Fig. 2a, c . The results after 200 folding dilution during LA tests are also depicted in Fig. 2b, d). PS of F2 and F9 after dilution were 57.7 and 12.8 nm, respectively, with PSD of 4.3 and 2.4, respectively. As the details show, F2 appears to be substantially influenced by dilution, while negligible changes are observed in F9 after dilution.

Fig. 2
figure 2

a F2 formulation before dilution. b F2 formulation after dilution. c F9 formulation before dilution. d F9 formulation after dilution

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Comparison between LA of Dill EO and the two selected nanoemulsions (F2 and F9) are depicted in Fig. 3. Samples without oil had no larvicidal effect (data not shown). LA of F9 was significantly better than the other two samples while no significant difference was observed between bulk Dill EO and F2.

Fig. 3
figure 3

Comparison of larvicidal activity of bulk Dill EO vs. its nanoemulsions (F2: unstable nanoemulsions after 200 times dilution during larvicide test, F9: stable nanoemulsion)

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This is for the first time that a nanoemulsion of Dill EO is reported and its LA is compared with its bulk form against one of the main malaria vectors, An. stephensi. Interestingly, LA of nanoemulsion which suffered negligible changes in structure after dilution (F9) showed significantly better LA compared with the other nanoemulsion (F2) with mortality %: 88.1 vs. 73.4. It is arguable that after dilution, by breaking nanostructure of F2, practically, no difference may be determined between F2 and bulk EO. Thus, similar LA is expected for F2 and bulk EO (see Figs. 2 and 3). However, in the case of F9, small size of the nanoemulsion (even after dilution) has caused a significant improvement in the efficacy. We believe this is the reason for the fact that in the literature only LA of nanoemulsions of EOs has been reported, and no comparison has been made between LA of nanoemulsions with that of bulk essential oil (da Rodrigues et al. 2014; Duarte et al. 2015; Oliveira et al. 2016). In other words, to prove the superior efficacy of nanoemulsions as larvicides, the effect of dilution on structure of nanoemulsion should be considered: a fact that appears to be neglected.

Characterization of selected nanoemulsion

Considering LA results, F9 was chosen as the optimum formulation. For investigation of its physical stability, the sample was stored at 4 °C and room temperature, for 30 days. No creaming or phase separation occurred, even after centrifugation at 25000 rpm. These results suggest proper stability for the preparation. TEM image of undiluted form of F9 is illustrated in Fig. 4. Its particles appear to be spherical with PS of ~11.1 nm.

Fig. 4
figure 4

TEM image of undiluted form of F9 formulation

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Conclusion

To obtain a nanoemulsion from essential oils with proper LA, concentration of the ingredients should be optimized to maintain structural stability of the nanoemulsion after 200-times dilution. Such a nanoemulsion is expected to show higher LA compared with bulk essential oil. This formulation can be suggested as low-cost, environment friendly larvicide, with activity comparable to industrial larvicides. The optimum formulation obtained in our work (i.e., F9) was a preparation with appropriate activity against larvae of An. stephensi with no minimum environmental effect.