# Larvicidal Effects of Nanoliposomes Containing Clove and Cinnamon Essential Oils, Eugenol, and Cinnamaldehyde against the Main Malaria Vector,Anopheles stephensi Liston

Authors: Alireza Sanei-Dehkordi; Roghayeh Heiran; Ghazaal Roozitalab; Narges Elahi; Mahmoud Osanloo
Journal: Psyche: A Journal of Entomology
 (2022)

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## Abstract

The use of larvicides, especially in endemic regions, is recommended for malaria control. However, due to the excessive use of synthetic larvicides, resistance in mosquitoes and environmental pollution have been challenges. In the current study, nanoliposome containing clove and cinnamon essential oils and their major ingredients, i.e., eugenol and cinnamaldehyde, were first prepared; particle size and successful loading were investigated using DLS (Dynamic Light Scattering) and ATR-FTIR (Attenuated Total Reflection-Fourier Transform InfraRed) analysis. Larvicidal effects of the nanoliposomes and nonformulated samples were then investigated againstAnopheles stephensi. The best-observed efficacy (LC50 5.4 μg/mL) was related to nanoliposomes containing eugenol with a particle size of 109 ± 4 nm. However, LC50 values of the other three nanoformulations were also around 10 μg/mL; all four prepared nanoformulations were thus introduced as natural larvicides for further investigations in the field conditions.


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## Body

## 1. Introduction

Mosquitoes (Diptera: Culicidae) transmit malaria, dengue, yellow fever, encephalitis, filariasis, chikungunya, and Zika virus [1, 2]. Around 30 species from 400 identified Anopheles mosquito species are the vector of malaria to humans [3]. Anopheles stephensi Liston. is one of the most important malaria vectors in the Middle East and South Asia; however, it has recently expanded to Ethiopia, Djibouti, Lakshadweep, and Sri Lanka [4, 5]. Larvae are the weakest members in the life cycle of mosquitoes; the use of larvicides is thus recommended to control malaria transmission, especially in endemic regions [6, 7]. However, the excessive use of synthetic larvicides has led to widespread resistance or intolerance, adverse environmental risks, and side effects on human health or other nontarget species [8, 9].Aromatic plants generate secondary metabolites known as essential oils (EOs), with various biological effects such as larvicidal and repellent effects. For instance,Syzygium aromaticum (L.) Merr. & L.M.Perry (clove) and Cinnamomum zeylanicum Blume (cinnamomum) are two medicinally important plants; their EOs possess larvicidal effects [10, 11]. The EOs with distinct properties such as eco-compatibility, biodegradability, and biocompatibility are proposed as proper alternatives for synthetic ones [12, 13]. However, the use of EOs as larvicides is hampered by their water immiscibility, high volatility, heating, swift oxidation, and degradation of on-air exposure [14, 15]. The preparation of EO-based nanoformulations has been recently proposed to meet the challenges [16]. Liposomes are minute vesicles comprising a lipid bilayer of amphiphilic molecules mimicking cells [17, 18]. Cargoes such as EOs or other natural larvicides could be entrapped into nanoliposomes to enhance stability, potency, efficacy, and durability [19, 20].This study first investigated the larvicidal effects of clove and cinnamomum EOs and their major ingredients (eugenol and cinnamaldehyde) againstA. stephensi. Then, an attempt was made to improve their efficacy by preparing nanoliposomes containing each.


## 2. Materials and Methods

### 2.1. Materials

All commercially available compounds were used as received. Wool fat cholesterol, tween 20, egg yolk lecithin, cinnamaldehyde, eugenol, and absolute ethanol were obtained from Merck Chemicals Co. (Germany). Cinnamon and clove EOs were purchased from Zardband Pharmaceuticals Co. and Green Plants of Life Co. Ltd. (Iran), companies with proprietary areas to grow the medicinal plants. This research used the late third and early fourth instar larvae ofA. stephensi (Bandar-e-Abbas strain); they were supplied from the Hormozgan University of Medical Sciences (Iran). All colonies were reared and maintained under the recommended conditions; 27 ± 2°C, 65 ± 5% relative humidity, in 12L : 12D h photoperiod (L: light, D: dark). We used the polytetrafluoroethylene (PTFE)-based membrane method for blood-feeding adult female mosquitoes [21].


### 2.2. Preparation of Loaded Nanoliposomes

The nanoliposomes containing EOs, eugenol, and cinnamaldehyde were prepared by the ethanol injection method [22]. The process of preparing loaded nanoliposomes is illustrated in Figure 1. Lecithin (3% w/v), cholesterol (1.0% w/v), tween 20 (0.5%), and each of eugenol, clove EO, cinnamaldehyde, and cinnamon EO (2.0% w/v) was first fully dissolved in absolute ethanol at room temperature overnight (2000 rpm). After that, 1 mL of the obtained mixture was added dropwise to 4 mL of distilled water (2000 rpm). The mixture was kept under stirring conditions for 40 minutes to stabilize formed nanoliposomes. The prepared samples were abbreviated as eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo.Figure 1
Preparation of nanoliposome containing eugenol, clove EO, cinnamaldehyde, and cinnamon EO.


### 2.3. Size Characterization

The mean diameter and particle size distribution (SPAN) of all nanoliposomes were investigated using a dynamic light scattering (DLS) instrument (K-One Nano, Ltd, Korea). In addition, the SPAN of the samples was also calculated by the equation d90—d 10/d50. Where d is diameter and 90, 10, and 50 are percentile of particles with lower diameter than these values.


### 2.4. Investigation of Loading of EOs, Eugenol, and Cinnamaldehyde in the Nanoliposomes

The Attenuated Total Reflection-Fourier Transform InfraRed (ATR-FTIR) was investigated to investigate the successful loading of eugenol, clove EO, cinnamaldehyde, and cinnamon EO into nanoliposome. Before being subjected to the analysis, the free nanoliposome and each loaded one was centrifuged for 60 min at 12000 g (4°C). The obtained pellets were stored at room temperature for three days to reduce their moisture. The spectra of each sample in the 400 to 4000 cm−1 were then recorded by a spectroscopy apparatus (Bruker Company, Model Tensor II, Germany).


### 2.5. Larvicidal Bioassays

Eugenol, clove EO, cinnamaldehyde, and cinnamon EO were dissolved in ethanol at a concentration of 2.0% w/v, equal to the concentration of the prepared nanoliposomes. Larvicidal effects of nonformulated and nanoformulated samples (eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo) were investigated in line with the WHO guidelines [9]. Briefly, by adding different amounts (31.3, 62.5, 125, 250, 500, and 1000 μL) of the samples to batches of A. stephensi larvae (25 n in 200 mL dechlorinated water), their concentration was fixed at 100, 50, 25, 12.50, 6.25, and 3.13 μg/mL. After 24 h exposure, larval mortality was calculated; larvae with no response to stimulation with a probe were considered dead.


### 2.6. Statistical Analyses

Larvicidal bioassay was carried out in triplicate, and larval mortality was presented as mean ± standard deviation. Calcusyn software (free version, BIOSOFT Co., UK) was used to calculate LC50 values of all samples with their upper and lower confidence interval (95%). The nonoverlap between the samples’ upper and lower limit values was interpreted as statistically significant.


## 2.1. Materials

All commercially available compounds were used as received. Wool fat cholesterol, tween 20, egg yolk lecithin, cinnamaldehyde, eugenol, and absolute ethanol were obtained from Merck Chemicals Co. (Germany). Cinnamon and clove EOs were purchased from Zardband Pharmaceuticals Co. and Green Plants of Life Co. Ltd. (Iran), companies with proprietary areas to grow the medicinal plants. This research used the late third and early fourth instar larvae ofA. stephensi (Bandar-e-Abbas strain); they were supplied from the Hormozgan University of Medical Sciences (Iran). All colonies were reared and maintained under the recommended conditions; 27 ± 2°C, 65 ± 5% relative humidity, in 12L : 12D h photoperiod (L: light, D: dark). We used the polytetrafluoroethylene (PTFE)-based membrane method for blood-feeding adult female mosquitoes [21].


## 2.2. Preparation of Loaded Nanoliposomes

The nanoliposomes containing EOs, eugenol, and cinnamaldehyde were prepared by the ethanol injection method [22]. The process of preparing loaded nanoliposomes is illustrated in Figure 1. Lecithin (3% w/v), cholesterol (1.0% w/v), tween 20 (0.5%), and each of eugenol, clove EO, cinnamaldehyde, and cinnamon EO (2.0% w/v) was first fully dissolved in absolute ethanol at room temperature overnight (2000 rpm). After that, 1 mL of the obtained mixture was added dropwise to 4 mL of distilled water (2000 rpm). The mixture was kept under stirring conditions for 40 minutes to stabilize formed nanoliposomes. The prepared samples were abbreviated as eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo.Figure 1
Preparation of nanoliposome containing eugenol, clove EO, cinnamaldehyde, and cinnamon EO.


## 2.3. Size Characterization

The mean diameter and particle size distribution (SPAN) of all nanoliposomes were investigated using a dynamic light scattering (DLS) instrument (K-One Nano, Ltd, Korea). In addition, the SPAN of the samples was also calculated by the equation d90—d 10/d50. Where d is diameter and 90, 10, and 50 are percentile of particles with lower diameter than these values.


## 2.4. Investigation of Loading of EOs, Eugenol, and Cinnamaldehyde in the Nanoliposomes

The Attenuated Total Reflection-Fourier Transform InfraRed (ATR-FTIR) was investigated to investigate the successful loading of eugenol, clove EO, cinnamaldehyde, and cinnamon EO into nanoliposome. Before being subjected to the analysis, the free nanoliposome and each loaded one was centrifuged for 60 min at 12000 g (4°C). The obtained pellets were stored at room temperature for three days to reduce their moisture. The spectra of each sample in the 400 to 4000 cm−1 were then recorded by a spectroscopy apparatus (Bruker Company, Model Tensor II, Germany).


## 2.5. Larvicidal Bioassays

Eugenol, clove EO, cinnamaldehyde, and cinnamon EO were dissolved in ethanol at a concentration of 2.0% w/v, equal to the concentration of the prepared nanoliposomes. Larvicidal effects of nonformulated and nanoformulated samples (eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo) were investigated in line with the WHO guidelines [9]. Briefly, by adding different amounts (31.3, 62.5, 125, 250, 500, and 1000 μL) of the samples to batches of A. stephensi larvae (25 n in 200 mL dechlorinated water), their concentration was fixed at 100, 50, 25, 12.50, 6.25, and 3.13 μg/mL. After 24 h exposure, larval mortality was calculated; larvae with no response to stimulation with a probe were considered dead.


## 2.6. Statistical Analyses

Larvicidal bioassay was carried out in triplicate, and larval mortality was presented as mean ± standard deviation. Calcusyn software (free version, BIOSOFT Co., UK) was used to calculate LC50 values of all samples with their upper and lower confidence interval (95%). The nonoverlap between the samples’ upper and lower limit values was interpreted as statistically significant.


## 3. Results

### 3.1. Particles Size of the Prepared Nanoliposomes

DLS profiles of the eugenol-lipo and clove-lipo with particle sizes of 109 ± 4 and 158 ± 4 nm are depicted in Figure2. Besides, their SPAN values were calculated as 0.96 and 0.96. Moreover, particle sizes of the cinnamaldehyde-lipo and cinnamon-lipo were obtained as 111 ± 6 and 195 ± 9 nm, and SPAN values were 0.96 and 0.97 (Figure 3). SPAN values of all mentioned formulations were lower than 1, so their narrow particle size distribution was confirmed [23].Figure 2
DLS profile of (a) eugenol-lipo 109 ± 4 nm (SPAN 0.96), (b) clove-lipo 158 ± 4 nm (SPAN 0.96).
(a)(b)Figure 3
DLS profile of (a) cinnamaldehyde-lipo 111 ± 6 nm (SPAN 0.96) (b) cinnamon-lipo 195 ± 9 nm (SPAN 0.97).
(a)(b)


### 3.2. Confirming Loading in the Liposomes

ATR-FTIR spectroscopy is one of the most useful methods to detect whether the EOs or major compounds were successfully incorporated into the liposome. The ATR-FTIR spectra of free liposome (Figure4(a)), eugenol-lipo (Figure 4(b)), clove-lipo (Figure 4(c)), cinnamaldehyde-lipo (Figure 4(d)), and cinnamon-lipo (Figure 4(e)) are shown in Figure 4.Figure 4
ATR-FTIR analyses of (a) free liposomes, (b) eugenol-lipo, (c) clove-lipo, (d) cinnamaldehyde-lipo, and (e) cinnamon-lipo.The free liposome spectrum showed the C-H and C-O stretching modes at 2980–2904 and 1044 cm−1. The bands at 1453–1274 cm−1 were ascribed to the bending modes of CH2, CH3, and COH, and the absorption signals at 1274–877 cm−1 were attributed to C-N and PO bonds phospholipid.From the eugenol-lipo spectrum (Figure4(b)), the hydroxy stretching (liposome and eugenol) at around 3377 cm−1 and C-H stretching (liposome and eugenol) at 3004, 2923, and 2852 cm−1 could be clearly observed. The band ascribed to the stretching vibration of the carbonyl group of liposomes observed at 1710 cm−1, and the absorptions at 1638, 1612, 1513, and 1464 cm−1 were attributed to C=C stretching vibrations. The sharp band at 1513 cm−1 was assigned to aromatic C=C stretching of eugenol. The bending mode of CH2 and CH3 appeared at 1431 and 1367 cm−1, and the new absorption bands at 1267–1034 cm−1 were attributed to C-O stretching modes of eugenol. Moreover, the bending modes of CH and C=C and the vibrations of C-N, P=O, and P-O were observed at 1367–647 cm−1. From the results, we could confirm the loading of eugenol into liposomes.Nanoliposome with the addition of clove EO (clove-lipo) exhibited similar major peaks of liposome and clove EO (Figure4(c)). The absorption bands of eugenol were prominent in the spectrum of clove-lipo. The broadband at 3350 cm−1 and the bands at 3004, 2923, and 2852 cm−1 signified O-H and C-H stretch. The bands at 1732 and 1712 cm−1 correspond to carbonyl groups, the strong band at 1514 cm−1 is due to aromatic C=C absorption, and the other C=C vibrations appeared at 1638–1463 cm−1. The bands at 1432 and 1366 cm−1 result from the bending vibration of CH2 and CH3. The wavenumbers of 1268–1037 cm−1 represented the vibrations of C-O and C-N bonds, and the absorption of P-O and P=O appeared at 1268–721 cm−1. Overall, the results indicated that clove EO was successfully loaded into liposomes.When cinnamaldehyde was incorporated into the liposome (Figure4(d)), the spectra represented peaks at 3390 and 1708 cm−1 related to hydroxy and carbonyl moieties, which is the characteristic peak of the liposome. The strong peak at 2922 cm−1 approved the presence of CH bonds in liposome and cinnamaldehyde, CO-H, and carbonyl stretching vibrations of cinnamaldehyde were located at 2851 and 1676 cm−1, respectively. The characteristic peaks at 1676–1372 cm−1 were attributed to stretching modes of C=C, and bending modes of CH2 and CH3 groups in cinnamaldehyde and liposome. The bands between 1296-1056 cm−1 indicated the presence of C-O (both EO and liposome) and PO2− bonds (in liposome), while the vibration of (CH3)3N+ was located around 952 and 892 cm−1, respectively. All of which confirm the existence of cinnamaldehyde in the liposome.The ART-FTIR characteristic peaks for cinnamon-lipo have been illustrated in Figure4(e). A broad peak around 3338 cm−1 showed the presence of hydroxy groups for alcoholic and phenolic hydroxy groups of cinnamon fractions and liposomes. The band at 2923 cm−1 indicates a C-H stretch in both cinnamon and liposome, and the absorbance bands at 2852 and 2739 cm−1 revealed the presence of the CO-H bond for aldehydes. The peak at 1730 cm−1 was recognized due to the carbonyl group of liposomes. Besides this, the peaks at 1675 and 1625 cm−1 may be attributed to the stretching vibration of carbonyl groups corresponding to cinnamaldehyde and other aldehydes in cinnamon EO. Furthermore, the bands between 1606-1377 cm−1 correspond to C=C stretching modes and C-H bending modes of methyl and methylene moieties. The strong absorption at 1121 cm−1 was due to the stretch of C-O bonds, and the other characteristic peaks from 1328 to 688 cm−1 were assigned for vibrations of C-N, C-O, P=O, and P-O bonds, deformation of COH, bending modes of CH, as well as the long-chain band.


#### 3.2.1. Larvicidal Effects of the Samples

Larvicidal effects of free liposomes at different amounts in the larvicidal test are depicted in Figure5. These amounts were equal to the values used to reach the examined concentrations of the samples containing the EO (eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo); only 8% of larvae survival was reduced at the highest amount (1 mL). Besides, larvicidal effects of all samples, including nonformulated samples and nanoformulations at a concentration range of 0–100 μg/mL, are shown in Figure 6. Interstingly, eugenol-lipo at 12.5–100 μg/mL concentrations showed perfect efficacy (caused 100% larvicidal effect).Figure 5
Larvicidal effects of free liposomes at different amounts in the larvicidal tests.Figure 6
Larvicidal effects of samples at different concentrations againstA. stephensi.Furthermore, obtained LC50 values of samples against A. stephensi are shown in Figure 7. Eugenol-lipo with an LC50 value of 5.37 (3.2–8.8) μg/mL showed the best efficacy; its LC50 was significantly more potent (p<0.05) than eugenol, clove EO, cinnamaldehyde, cinnamon-lipo, and cinnamon EO. Besides, LC50 values of three other nanoformulations, including clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo, were obtained as 10.5 (6.2–17.9), 9.8 (5.6–17.1), and 13.7 (9.3–20.3) μg/mL. Moreover, LC50 values of nonformulated samples, including eugenol, clove EO, cinnamaldehyde, and cinnamon EO, were observed as 67.6 (55.3–82.6), 57.7 (53.6–62.1), and 62.2 (61.4–63.1) μg/mL.Figure 7
Obtained LC50 value of samples against A. stephensi; ∗∗p<0.05.


## 3.1. Particles Size of the Prepared Nanoliposomes

DLS profiles of the eugenol-lipo and clove-lipo with particle sizes of 109 ± 4 and 158 ± 4 nm are depicted in Figure2. Besides, their SPAN values were calculated as 0.96 and 0.96. Moreover, particle sizes of the cinnamaldehyde-lipo and cinnamon-lipo were obtained as 111 ± 6 and 195 ± 9 nm, and SPAN values were 0.96 and 0.97 (Figure 3). SPAN values of all mentioned formulations were lower than 1, so their narrow particle size distribution was confirmed [23].Figure 2
DLS profile of (a) eugenol-lipo 109 ± 4 nm (SPAN 0.96), (b) clove-lipo 158 ± 4 nm (SPAN 0.96).
(a)(b)Figure 3
DLS profile of (a) cinnamaldehyde-lipo 111 ± 6 nm (SPAN 0.96) (b) cinnamon-lipo 195 ± 9 nm (SPAN 0.97).
(a)(b)


## 3.2. Confirming Loading in the Liposomes

ATR-FTIR spectroscopy is one of the most useful methods to detect whether the EOs or major compounds were successfully incorporated into the liposome. The ATR-FTIR spectra of free liposome (Figure4(a)), eugenol-lipo (Figure 4(b)), clove-lipo (Figure 4(c)), cinnamaldehyde-lipo (Figure 4(d)), and cinnamon-lipo (Figure 4(e)) are shown in Figure 4.Figure 4
ATR-FTIR analyses of (a) free liposomes, (b) eugenol-lipo, (c) clove-lipo, (d) cinnamaldehyde-lipo, and (e) cinnamon-lipo.The free liposome spectrum showed the C-H and C-O stretching modes at 2980–2904 and 1044 cm−1. The bands at 1453–1274 cm−1 were ascribed to the bending modes of CH2, CH3, and COH, and the absorption signals at 1274–877 cm−1 were attributed to C-N and PO bonds phospholipid.From the eugenol-lipo spectrum (Figure4(b)), the hydroxy stretching (liposome and eugenol) at around 3377 cm−1 and C-H stretching (liposome and eugenol) at 3004, 2923, and 2852 cm−1 could be clearly observed. The band ascribed to the stretching vibration of the carbonyl group of liposomes observed at 1710 cm−1, and the absorptions at 1638, 1612, 1513, and 1464 cm−1 were attributed to C=C stretching vibrations. The sharp band at 1513 cm−1 was assigned to aromatic C=C stretching of eugenol. The bending mode of CH2 and CH3 appeared at 1431 and 1367 cm−1, and the new absorption bands at 1267–1034 cm−1 were attributed to C-O stretching modes of eugenol. Moreover, the bending modes of CH and C=C and the vibrations of C-N, P=O, and P-O were observed at 1367–647 cm−1. From the results, we could confirm the loading of eugenol into liposomes.Nanoliposome with the addition of clove EO (clove-lipo) exhibited similar major peaks of liposome and clove EO (Figure4(c)). The absorption bands of eugenol were prominent in the spectrum of clove-lipo. The broadband at 3350 cm−1 and the bands at 3004, 2923, and 2852 cm−1 signified O-H and C-H stretch. The bands at 1732 and 1712 cm−1 correspond to carbonyl groups, the strong band at 1514 cm−1 is due to aromatic C=C absorption, and the other C=C vibrations appeared at 1638–1463 cm−1. The bands at 1432 and 1366 cm−1 result from the bending vibration of CH2 and CH3. The wavenumbers of 1268–1037 cm−1 represented the vibrations of C-O and C-N bonds, and the absorption of P-O and P=O appeared at 1268–721 cm−1. Overall, the results indicated that clove EO was successfully loaded into liposomes.When cinnamaldehyde was incorporated into the liposome (Figure4(d)), the spectra represented peaks at 3390 and 1708 cm−1 related to hydroxy and carbonyl moieties, which is the characteristic peak of the liposome. The strong peak at 2922 cm−1 approved the presence of CH bonds in liposome and cinnamaldehyde, CO-H, and carbonyl stretching vibrations of cinnamaldehyde were located at 2851 and 1676 cm−1, respectively. The characteristic peaks at 1676–1372 cm−1 were attributed to stretching modes of C=C, and bending modes of CH2 and CH3 groups in cinnamaldehyde and liposome. The bands between 1296-1056 cm−1 indicated the presence of C-O (both EO and liposome) and PO2− bonds (in liposome), while the vibration of (CH3)3N+ was located around 952 and 892 cm−1, respectively. All of which confirm the existence of cinnamaldehyde in the liposome.The ART-FTIR characteristic peaks for cinnamon-lipo have been illustrated in Figure4(e). A broad peak around 3338 cm−1 showed the presence of hydroxy groups for alcoholic and phenolic hydroxy groups of cinnamon fractions and liposomes. The band at 2923 cm−1 indicates a C-H stretch in both cinnamon and liposome, and the absorbance bands at 2852 and 2739 cm−1 revealed the presence of the CO-H bond for aldehydes. The peak at 1730 cm−1 was recognized due to the carbonyl group of liposomes. Besides this, the peaks at 1675 and 1625 cm−1 may be attributed to the stretching vibration of carbonyl groups corresponding to cinnamaldehyde and other aldehydes in cinnamon EO. Furthermore, the bands between 1606-1377 cm−1 correspond to C=C stretching modes and C-H bending modes of methyl and methylene moieties. The strong absorption at 1121 cm−1 was due to the stretch of C-O bonds, and the other characteristic peaks from 1328 to 688 cm−1 were assigned for vibrations of C-N, C-O, P=O, and P-O bonds, deformation of COH, bending modes of CH, as well as the long-chain band.


### 3.2.1. Larvicidal Effects of the Samples

Larvicidal effects of free liposomes at different amounts in the larvicidal test are depicted in Figure5. These amounts were equal to the values used to reach the examined concentrations of the samples containing the EO (eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo); only 8% of larvae survival was reduced at the highest amount (1 mL). Besides, larvicidal effects of all samples, including nonformulated samples and nanoformulations at a concentration range of 0–100 μg/mL, are shown in Figure 6. Interstingly, eugenol-lipo at 12.5–100 μg/mL concentrations showed perfect efficacy (caused 100% larvicidal effect).Figure 5
Larvicidal effects of free liposomes at different amounts in the larvicidal tests.Figure 6
Larvicidal effects of samples at different concentrations againstA. stephensi.Furthermore, obtained LC50 values of samples against A. stephensi are shown in Figure 7. Eugenol-lipo with an LC50 value of 5.37 (3.2–8.8) μg/mL showed the best efficacy; its LC50 was significantly more potent (p<0.05) than eugenol, clove EO, cinnamaldehyde, cinnamon-lipo, and cinnamon EO. Besides, LC50 values of three other nanoformulations, including clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo, were obtained as 10.5 (6.2–17.9), 9.8 (5.6–17.1), and 13.7 (9.3–20.3) μg/mL. Moreover, LC50 values of nonformulated samples, including eugenol, clove EO, cinnamaldehyde, and cinnamon EO, were observed as 67.6 (55.3–82.6), 57.7 (53.6–62.1), and 62.2 (61.4–63.1) μg/mL.Figure 7
Obtained LC50 value of samples against A. stephensi; ∗∗p<0.05.


## 3.2.1. Larvicidal Effects of the Samples

Larvicidal effects of free liposomes at different amounts in the larvicidal test are depicted in Figure5. These amounts were equal to the values used to reach the examined concentrations of the samples containing the EO (eugenol-lipo, clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo); only 8% of larvae survival was reduced at the highest amount (1 mL). Besides, larvicidal effects of all samples, including nonformulated samples and nanoformulations at a concentration range of 0–100 μg/mL, are shown in Figure 6. Interstingly, eugenol-lipo at 12.5–100 μg/mL concentrations showed perfect efficacy (caused 100% larvicidal effect).Figure 5
Larvicidal effects of free liposomes at different amounts in the larvicidal tests.Figure 6
Larvicidal effects of samples at different concentrations againstA. stephensi.Furthermore, obtained LC50 values of samples against A. stephensi are shown in Figure 7. Eugenol-lipo with an LC50 value of 5.37 (3.2–8.8) μg/mL showed the best efficacy; its LC50 was significantly more potent (p<0.05) than eugenol, clove EO, cinnamaldehyde, cinnamon-lipo, and cinnamon EO. Besides, LC50 values of three other nanoformulations, including clove-lipo, cinnamaldehyde-lipo, and cinnamon-lipo, were obtained as 10.5 (6.2–17.9), 9.8 (5.6–17.1), and 13.7 (9.3–20.3) μg/mL. Moreover, LC50 values of nonformulated samples, including eugenol, clove EO, cinnamaldehyde, and cinnamon EO, were observed as 67.6 (55.3–82.6), 57.7 (53.6–62.1), and 62.2 (61.4–63.1) μg/mL.Figure 7
Obtained LC50 value of samples against A. stephensi; ∗∗p<0.05.


## 4. Discussions

Chemical compositions of the used clove and cinnamon EOs in the current study have been investigated in our previous studies using Gas Chromatography-Mass Spectrometry analysis. As a result, thirty-three constituents were identified in clove EO; eugenol (65.41%),trans-caryophyllene (12.06%), eugenol acetate (9.85%), caryophyllene oxide (3.00%), and α-humulene (1.73%) were its five major constituents [24]. Besides, thirty constituents were identified in cinnamon EOs with five major compounds, including cinnamaldehyde (62.04%), linalool (6.96%), trans-caryophyllene (6.60%), trans-cinnamyl acetate (4.29%), and benzyl benzoate (3.32%) [25].Nanoliposomes are tiny spherical vesicles (diameter <200 nm) spontaneously formed by phospholipids bilayer membrane in an aqueous medium [26, 27]. Hydrophobic materials such as eugenol and cinnamaldehyde are more loaded in the membrane; however, EOs (e.g., clove and cinnamon), a mixture of hydrophobic/hydrophilic substances, are loaded both in the membrane and central aqueous cavity [17, 19]. In the current study particle size of the liposomes containing eugenol and cinnamaldehyde was smaller than the liposomes containing clove and cinnamon EO, probably due to the loading of larger amounts EOs’ compounds into the central cavity.The loading process improves the physicochemical stability of the cargoes as pesticides and prevents the degradation of active agents [28, 29]. In addition, nanocarriers containing cargo provide a controlled release at the site of action, and thus their efficacy periods are longer [30, 31]. Moreover, particles with nanoscale allowed entering larvae bodies pores; consequently, the spreading of cargoes improves [32, 33]. Besides, when a solute such as EO is dissolved in a solvent, its droplet size is less than ∼1 nm [34, 35]. Therefore, inside a nanoparticle with a diameter of 200 nanometers, around 1 million drops could be loaded; the packages containing EO reach the larval body [16, 36]. As a result, the bioactivity and efficacy of EO-based nanoformulations are generally higher than those of free EOs [37, 38].In the current study, LC50 values of eugenol and cinnamaldehyde were improved compared with their nanoliposomal states 12 and 6 folds. This improvement for clove and cinnamon EOs versus liposomes containing them was observed as 6 and 2 folds. Reviewing the literature, size-dependent improvements are not limited to the current study. For instance, the larvicidal effects of limonene and three limonene reach EOs from the citrus family, including C. aurantium, C. limon, and C. sinensis against A. stephensi were investigated; LC50 values were obtained as 20, 62, 13, and 12 μg/mL. Their nanoliposomal state with LC50 values of 13, 6, 6, and 9 was significantly more potent than the nonformulated states [22]. In another paper, larvicidal effects of carvacrol and two carvacrol reach EOs, including Satureja khuzestanica and Zataria multiflora EO against A. stephensi, were investigated; LC50 values were obtained as 128, 42, and 79 μg/mL. However, their nanoliposomal states with LC50 values of 11, 12, and 10 μg/mL were significantly more potent than nonformulated states [39]. Interestingly, the potency of eugenol-lipo with an LC50 value of 5 μg/mL against A. stephensi was more potent than the mentioned reports.Furthermore, the LC50 value of chitosan nanoparticles containing cinnamon EO was obtained as 2.98 μg/mL in our previous study [11]; however, the LC50 value of nanoliposomes containing cinnamon EO was achieved at 13.7 μg/mL in the current study. Besides, the larvicidal effect of free chitosan nanoparticles in the mentioned report and another report by our team were around 18% [11, 40]. While free liposomes in the current study did not show a significant larvicidal effect, one of the differences in the result of that study with the present study is thus the difference in the nanocarriers used. Moreover, liposomes have an advantage over chitosan nanoparticles due to their high loading capacity for EO or their main constituents. However, more research is needed at the same time for these carriers.


## 5. Conclusions

The current study used eugenol, clove EO, cinnamaldehyde, and cinnamon EOs as natural larvicides against the main malaria vector mosquito,A. stephensi. An attempt was made to improve their efficacy by preparing nanoliposomes containing each; interestingly, their efficacy (LC50 values∼10 μg/mL) about 2–12 folds was improved. The prepared nanoliposomes were introduced as natural larvicides for further investigations and against other medically critical mosquitoes.



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*Source: 9991238-2022-09-16.xml*