Evaluation of larvicidal enhanced activity of sandalwood oil via nano-emulsion against Culex pipiens and Ades aegypti

Graphical abstract


Introduction
Mosquitoes are important medical pests given their role in transmitting diseases among humans or animals. Vector control is the primary way to reduce public concerns about mosquitoborne diseases including filariasis, dengue fever, malaria, and leishmaniasis (Wilson et al., 2020). The control of larval stages of mosquito considered more efficient way to reduce the spread of mosquitoes than that of adults (WHO, 2013).
The search for environmentally friendly alternatives, like plants or oils, rich in secondary metabolites is a modern trend because of their efficiency, minimal toxicity, biodegradability, and the capability to reduce resistance (S ßengül Demirak and Canpolat, 2022).
Nanotechnology is a multidisciplinary science that entails creating and using different systems and structures at the nanometer scale. Several forms of nano-emulsions, which are dispersed systems consisting of immiscible liquids and stabilizers, have been applied (McClements, 2012). Nano-emulsions are characterized by their thermodynamic stable and small droplets in size range 20-200 nm, leading to high efficacy (Jaiswal et al., 2015).
Sandalwood oil, with scientific name, Santalum album L., Family: Santalaceae, a product of the wood and roots of sandalwood tree, is an essential oil widely found in India and East Asian countries, as well as in the northern coast of Australia and Hawaiian island. Sandalwood tree is expensive worldwide as its products are used all over the world due to its great economic importance. Sandalwood essential oil was identified to contain >150 terpenoid compounds, majority of which are a and b-santalol components, as well as others minor components including a-santalene, bsantalene, and a-bergamotene (Zhang et al., 2019). The oil, as well as the main compounds, have low toxicity upon oral and dermal exposure in experimental animals and showed antioxidant and anti-inflammatory effects, reflecting its protective activity in a cerebral ischemia mouse model (Younis and Mohamed, 2020).
This study was designed to evaluate the larvicidal efficacy of Santalum album oil and its nano-emulsion against Culex pipiens, and Aedes aegypti 3rd instar larvae, and determine their effect on the detoxifying enzymes activity in larval tissues. Oil constituents' determination via gas chromatography-mass spectrometry analysis and oil phenolic compounds determination via liquid chromatography coupled with electrospray ionization and tandem mass spectroscopy.

Oil nano-emulsion preparation
The oil in water nano-emulsion was prepared by mixing 5 ml of sandalwood oil (at 45°C) in 50 ml beaker contained 10 ml distilled water, 0.5 g sodium cholate, 0.5 g sodium glycocholate and 3.5 ml tween 20 (at 45°C), stirred with magnetic stirrer until a clear emulsion was obtained. The mixture was quenched gradually with 50% v/v water, then, emulsified via sonication for 10 min at 200 W. The nano-emulsion was subjected to freeze drying lyophilization using SP Virtis Advantage Pro Laboratory Benchtop Freeze-Dryer Lyophilizer, with sucrose as a cryoprotectant (Yuan et al., 2008;Gundewadi et al., 2018).

Characterization of the oil nano-emulsion
2.3.1. Particle size and surface charge using DLS and TEM analysis The hydrodynamic radius and surface charge were investigated via dynamic light scattering (DLS) to determine the particle size (mean diameter) and zeta potential to confirm stability and uniformity by polydispersity index (PDI) and surface charge using zeta sizer nano Zs analyzer Malvern Panalytical, UK. One mg Sample was dispersed in 10 ml deionized water (Yuan et al., 2008). Particle morphology examined using TEM (Joel-1400 Flash) on carbon coated copper grids (600 mesh). Images were captured using CCD camera (EMT), the accelerating voltage was 80 kV (Yuan et al., 2008;Gundewadi et al., 2018).

Differential scanning calorimetry (DSC)
Lyophilized sandalwood nano-emulsion (5 g) was used to investigate the thermal stability profile (DSC-60, Shimadzu, Japan). The sample placed in standard aluminum pans with temperature raized from 2 to 200°C covering the thermogram at 10°C/min (Ji et al., 2016).

Gas chromatography-mass spectrometry (GC-MS)
Chemical composition of oil was determined using gas chromatography-mass spectrometry as detailed previously by El-Kasem Bosly (2022).

Liquid chromatography coupled with electrospray ionization and tandem mass spectroscopy (LC-ESI-MS/MS)
Phenolic compounds in the sandalwood oil sample were performed using LC-ESI-MS/MS for the separation. An ExionLC AC HPLC system and SCIEX Triple Quad 5500 + MS/MS system equipped with an electrospray ionization for detection. The column, ZORBAX SB-C18 (4.6 Â 100 mm, 1.8 lm) was used. Two mobile phases, A: 0.1% formic acid in water and B: acetonitrile in programming mode as follows: 2% B from 0 to 1 min, 2 to 60% B from 1 to 21 min, 60% B from 21 to 25 min and 2% B from 25.01 to 28 min with 0.8 ml/min, as flow rate and the sample was 3 ll in volume. Positive and negative ionization modes were used in the same run in sittings for the multiple reactions monitoring (MRM) of the selected polyphenols as: curtain gas was 25 psi; for sitting positive and negative modes the IonSpray voltage were 4500 and-4500, respectively; source temperature was 400°C; ion source gas 1 and 2 were 55 psi with a declustering potential at 50 V; collision energy at 25 eV and collision energy spread was 10 V.

Culex pipiens and Aedes aegypti mosquito colonies
Mosquito larvae of Culex pipiens and Aedes aegypti were reared as detailed by El-Kasem Bosly (2022).

Larvicidal assay
Larvicidal activities of sandalwood essential oil and its nanoemulsion were conducted against Culex pipiens and Aedes aegypti 3rd instar larvae according to WHO (2005). Two milliliters of the oil was placed in 100 ml water containing 2% tween 20 and subjected magnetic stirring (CR302, UK). Also, 2 ml of the prepared nano-emulsion was ultrasonicated in 100 ml water for equal distribution. Concentrations were prepared from the aforementioned preparations at 62.5, 125, 250, 500, 1000 and 1500 ppm. Twentyfive larvae from Cx pipiens and/or Ae aegypti were subjected to every-one concentration in glass beakers (250 ml in volume) comprising 150 ml of dechlorinated water (aqueous suspension) at 27 ± 2°C, 70 ± 10% relative humidity and a 12:12 h light/dark photoperiod. The experiment was replicated five times for each concentration per extract and control group (solvent only treated). Larval mortalities were recorded after 24 and 48 h.

Larval preparation for biochemical assays
Third instar larvae of both species were exposed to oil and/or its nano-emulsion at the calculated LD 50 in three replicates, as well as the control group, according to the aforementioned conditions in the larvicidal assay. Larvae were collected and weighed after 48 h from each group and pooled from each replicate for body homogenization in distilled water 10% (w/v) under ice and via cooling centrifugation at 4°C for 15 min at 10000 rpm the supernatant was used for the biochemical assays.

Data analysis
Percentage larval mortality was calculated according to Abbott (1925). The larval control mortality was less than 5%, did not need correction according to the WHO, (2005) guidelines. Mortality and biochemical data resulting from all replicates were analyzed by one-way analysis of variance (ANOVA) to find the differences among the activity between each oil or nano-emulsion concentrations using the least significant difference test. Also, all replicates data were subjected to analysis for determination of the larval LC 50 , LC 90 , and LC 95 as well as chi-square values within confidence limits at 95% by using probit analysis and regression between logarithm bas 10 of oil concentration and probit values. Data analysis was done via IBM SPSS Statistics v22 -64 bit software with statistical significance at p < 0.05.

Characteristics of oil nano-emulsion
Sandalwood oil nano-emulsion was characterized via DLS, which revealed particle average size of 195.7 nm and PDI of 0.342 (Fig. 1A), confirming homogeneous, stable and uniform narrow distributed nanoparticles. Zeta potential was À20.1 mV (Fig. 1B). The nano-emulsion morphology detected using TEM is represented in Fig. 2(A-C), showing regular spherical particles with a size in the range of 112-196 nm. Scanning electron micrograph (SEM) of lyophilized sandalwood nano-emulsion using sucrose as cryo-protectant was predicted smooth spherical particle shape (Fig. 3). Meanwhile, DSC thermogram showed an endothermic melting peak at 181°C (Fig. 4).

Larvicidal activities
Larval mortality data are represented in Table 3. In sandalwood oil -exposed groups, after 24 h, Cx. pipiens and Ae aegypti larvae exhibited 100% mortality at a dose of 1500 ppm with LD 50, LD 90 and LD 95

Biochemical results
Biochemical results represented in Table 4, showing significant decrease in TP content and ALP and b esterase enzymes activities in Cx. pipiens and Ae. aegypti exposed to both treatments with significant lowering effect of nano-emulsion as compared to oil. Meanwhile, a esterase and GST enzymes activities showed significant increase upon both treatments as compared to corresponding controls. In addition, nano-emulsion exposed groups showed significant increase in a esterase and GST enzymes activities in Cx.
pipiens and Ae. aegypti groups comparing to the parallel values in oil exposed groups.

Discussion
In recent years, there has been a great interest from health authorities and organizations in the significance of vector-borne diseases at the global and regional levels since they continue to demonstrate a significant health threatening to the societies worldwide (WHO, 2017;Valenzuela et al., 2018). Mosquito-borne diseases represent the largest measure of this fear, that's because of the mosquitoes ability to transmit many medical and veterinary diseases, like, filariasis, malaria, dengue fever, Rift Valley fever, Lumpy skin, and others which negatively affects human health and causes clear economic losses (Al-Seghayer et al., 1999;Singh et al., 2019). In addition to this interest, research on mosquito control based natural alternative agents instead of synthetic pesticides, with a clear appreciation in the scientific and medical community, especially natural products derived from plants (S ßengül Demirak and Canpolat, 2022). Because of their capability to win the goal for reducing pests without harming the environment, essential oils within their chemical constituents, exerted beneficial effects and due to their lipophilic nature acquired the capability for crossing membranes and hence, exerts their toxicity activity towards insects, as well as their antimicrobial, antibacterial, antifungal, antiviral in line with their miscellaneous activities (Stephane and Jules, 2020).
The sandalwood oil nano-emulsion prepared in the present study characterized by Zeta potential was within range À30-30 mV associated with stable nano-emulsion systems and the negative value is necessary for droplet-droplet repulsion and enhanced nano-emulsion stability. In addition, the recorded small PDI that described the degree of particles distribution uniformity in the emulsion confirmed good homogeneity indication (Danaei et al., 2018;Gul et al., 2022). TEM findings agree with DLS data, however, the particle size determined using TEM was smaller than that detected using DLS due to the sensitivity of technique (Klang et al., 2012). The characteristics of the nano-emulsion were in agreement with previous studies (González et al., 2016;Firooziyan et al., 2021;Zamaniahari et al., 2022). Differential scanning calorimetry can be applied for the recognition of microsponges when loaded molecules are entrapped nearby. Melting, boiling, and/or sublimation points of the entrapped molecules generally change or disappear. According to this, the presented melt-ing peak was thought to be the effect of the sucrose cryoprotectant and no significant endothermic peak was observed for the sandalwood oil nano-emulsion that it was liquid at room temperature.
The larval mortality results confirmed the sandalwood oil effect previously identified against Ae. aegypti larvae (Amer and Mehlhorn, 2006). In addition to the efficient larvicidal predicted action against Cx pipiens, Ae aegypti and Aedes albopictus larvae that reportedly due to the toxicity of the oil constituents (Zhu et al., 2008). Another study showed significant repellant and insecticidal activities of sandalwood oil and its main active ingredients aand bsantalols against Aphis gossypii and suggested sandalwood oil and its main compounds for use as possible ecofriendly management against Aphis gossypii (Roh et al., 2015). Sandalwood oil showed a repellent activity for the parasitic mite, Varroa jacobsoni which invades and threatens honeybee colonies (Imdorf et al., 1999) and against Lycoriella mali, Sciarid flies, with modest activity reported (Choi et al., 2006). Besides, santalol showed activity against the spider mite Tetranychus urticae (Roh et al., 2012) acting as acaricidal and oviposition deterring. Furthermore, Indian sandalwood tree (S. Album L.) has benificial properties in inhibiting insects' growth due to its chemical properties (Shankaranarayana et al. 1980).
The study results, showed a significant larvicidal efficacy of the nano-emulsion as compared to that of the oil against both larvae, revealing the enhanced activity of the nano-emulsion in agreement with Duarte et al. (2015), who evaluated rosemary essential oil nano-emulsion and its potential larvicidal effect against Ae. aegypti larvae. Moreover, Mahran (2022) evidenced the larvicidal improvement of basil and cumin essential oils in their nano-emulsion formulations against Cx. pipiens larvae.
The sandalwood oil nanoemulsion also recorded significant decrement in total protein contents in the exposed species as compared to their concentrations in the oil exposed larvae and both treatments showed total protein significant decrement as compared to control value, which proposed for the synthesis microsomal detoxifying enzymes (Massoud et al., 2001). The total protein decrement was confirmed in previous studies (Koodalingam et al., 2012;Sugumar et al, 2014). Esterases and GST function as detoxification enzymes for endogenous and exogenous chemicals to eliminate or transform them to less toxic metabolites through different metabolic pathways. The alteration of enzymes throughout the oil compounds action besides the role of enzymes in metabolizing oil constituents was previously proposed (Intirach et al., 2019). Sandalwood oil larvicidal activity was proposed through its target for the detoxifying enzymes (Tong and Bloomquist, 2013) which increased larval sensitivity to tannins and generally for phenolic compounds, in accordance with the predicted sandalwood oil compounds with proposed mosquitocidal activity (Rey et al., 1999;Rey et al., 2001).   Sandalwood oil showed antiviral activity against herpes simplex virus type 1 (HSV-1) in a dose dependent manner and the activity was proposed via oil increment effect on cellular GST enzyme activity (Benencia and Courrèges, 1999). Noting that besterase activity decreased in the present results, which is often the reverse of a-esterase activity as a saver for the larvae from the oil constituents' toxicity. Also, could be because esterase proteins have different substrate specificities resulting in different active sites of the two esterases (Montella et al., 2012). The present results may support the involvement of that enzymes in the detoxification of sandalwood oil or its nanoemulsion in the tested larvae.
Essential oil nano-emulsion protects the oil against oxidation and controls its release and bioactivity by increasing the exposed area and providing the interaction of oil active compounds with their target, resulting in increased stability and shelf life, decreasing degradation due to environmental factors. These properties indicate their effectiveness compared to crude and even pure oil (da Silva et al., 2022). In previous study neem oil nano-emulsion showed effective larvicidal potency aginst Cx. quinquefasciatus 3rd instar larvae (Anjali et al., 2012). Balasubramani et al, (2017) showed the larvicidal activity advantage of Vitex negundo L. leaf essential oil nano-emulsion (particle size, 200 nm) against Ae. aegypti larvae as compared to that of the oil after 12 and 24 h. Firooziyan et al, (2021) reported increased larvicidal efficacy of Cinnamim zelanicum nano-emulsion against An. stephensi larvae compared to the essential oil. Similarly, Aeollanthus suaveolens Mart. leaves oil in the nano-emulsion formulation (particle size 126.73 nm and zeta potential À16.25 mV) evaluated larval toxicity Ae. aegypti larvae (Lopes Martins et al., 2021). Table 1 Chemical constituents of sandalwood essential oil by gas chromatography-mass spectrometer (GC-MS).

No
Molecular formula Chemical name Area (%) RT    Significance at 0.05 level between different superscripts within the same column of each treatment. SEM, standard error of the mean; LCL, lower confidence limit; UCL, upper confidence limit. (*) reflects significance within the same concentration level between the two treatments within the same column.

Conclusion
The study verified the enhanced larvicidal potential of sandalwood oil nano-emulsion against Cx. pipiens and Ae. aegypti mosquito larvae as compared to that of the oil as well as alterations in the detoxifying enzymes based on oil active ingredients. Although the rational use of sandalwood oil is limited as insecticide due to the coast, it is used in a wide range of applications in fragrance and medicinal usage. The insecticidal activity offers a variety of use as a pesticide and the nano-emulsion formulation adds extra stability and elevates its toxicity against the tested mosquito larvae. The study recommends sandalwood oil nanoemulsion as a safe and stable larvicide against Cx. pipiens and Ae. aegypti and more biochemical investigations are warranted to explore more larvicidal mode of action.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data expressed as means ± SEM. SEM, standard error of the mean. Significance (p > 0.05) between larval groups represented by (*) superscripts as compared to their corresponding control within the same column. Different subscripts indicated significance between treatments (between columns). TP, total protein; ALP, alkalinphosphatase; GST, glutathione S-transferase.