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Beni-Suef University Journal of Basic and Applied Sciences
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Synthesis and efficacy of cinnamon oil formulations and their sustainable release against common house mosquito larvae

Beni-Suef University Journal of Basic and Applied Sciences volume 12, Article number: 118 (2023) Cite this article

Abstract

Background

Control of mosquitoes is considered an essential public health priority. This study was designed to estimate the larvicidal activity of two formulations of Cinnamomum zeylanicum EO for controlling Culex pipiens larvae.

Results

The prepared formulations were a nanoemulsion of cinnamon (CNE), cinnamon (CN) alone and ordinary cinnamon essential oil mixed with sesame oil (CSO). The cinnamon + sesame oil (CSO) was added as one part cinnamon to 3 parts SO. Different concentrations were prepared and applied following the WHO larvicidal bioassay protocol. Our findings revealed that the LC50 of the CNE form ranged from 85.3 µg/mL to 28.30 µg/mL. The LC50 of SO alone was 1265 µg/mL but when mixed with CNE to form the CSO mixture, this decreased to 159.00 µg/mL. In terms of residual effect, the ordinary form of cinnamon had a residual effect in water for 72 h at a dose of 1000 µg/ml, but this extended to 120 h at the same dose when the CNE form was used. However CSO did not have a residual effect, however.

Conclusion

The nanoemulsion form significantly improved the efficacy and residual effect of cinnamon against Culex pipiens larvae. Additionally, mixing cinnamon with sesame oil had a synergistic effect. This may assist control strategies against the house mosquito, Culex pipiens.

1 Background

Mosquitoes cause annoyance, and many diseases resulting from their bites, such as malaria, dengue fever, yellow fever, West Nile, chikungunya, Zika, and filariasis, makes them one of the globe’s deadliest pests [1]. Control of mosquitoes is therefore considered an essential public health priority. Malaria is transmitted by Anopheline mosquitos and causes an estimated 219 million cases worldwide, with over 400,000 deaths each year. Furthermore, Aedes mosquitos carry dengue illness. More than 3.9 billion individuals are at risk of infection, with an estimated 96 million symptomatic cases and 40,000 deaths per year [2].

Although manufactured insecticides are widely used to control mosquitoes, these can have negative impacts on the environment, and on human and animal health. In particular, insecticides can leave highly toxic and long-lasting residues in the environment and encourage the development of resistance in the target insects [3, 4].

Plant essential oils are secondary metabolites and are recognized as a safe and natural alternative source of bioactive substances suitable for the control of mosquitoes [5]. The essential oils act as nerve agents disrupting insects’ respiratory systems [6]. They can also be used as fumigants [7], repellents [8], antifeedants [9] toxins [10], and to interfere with insect growth, development, metamorphosis, and reproduction [11].

Cinnamon is a tropical tree related to the Lauraceae family; it is a tropical tree growing Sri Lanka, East and Middle Asia [12]. Cinnamon extract contains a range of bioactive phytochemicals, includes cinnamaldehyde, cinnamic acid, cinnamate, cinnamyl acetate, trans-cinnamaldehyde, and eugenol [13]. Various studies have reported that cinnamon essential oils have larvicidal, ovicidal, adulticidal, and repellent activities against mosquitoes [14].

Sesame (Sesamum indicum L.), meanwhile has been grown by humans for thousands of years. The essential oil crop is derived mainly from plants in the Pedaliaceae family, genus Sesamum, which is cultivated largely in Africa and, to a lesser extent, India [15]. Sesamum indicum peel extract has been shown to have larvicidal and repellent activities against Culex pipiens [16], Sesame oil has been identified as a good antioxidant that works synergistically with pesticides against Spodoptera littoralis [17]. Furthermore, an 8:2 blend of clove oil andsesame oil has been shown to possess a synergistic effect when applied against Callosobruchus maculatus (a common agricultural pest) [18].

Nanotechnology has recently attracted considerable attention as a method to maximize the effectiveness of botanical-based pesticides. [19, 20]. Benefits include the ability to focus drug delivery more directly to its place of action, lower doses, improved efficacy, and reduced side effects. Nanoemulsions can also preserve active compounds from degradation and deactivation, consequently prolonging the pesticide’s half-life [20]. Furthermore, the physicochemical qualities of nanoemulsions are better adapted to overcome the target organism's physiological barriers and can have better affinity with the target tissue. All in all, nanoemulsion techniques can double the efficacy of essential oils compared to their ordinary form [21].

In the above context, the main objective of this research was to assess the toxicity of two formulations of cinnamon essential oil against Culex pipiens larvae, with reference to its residual effect.

2 Methods

2.1 Source of the work compounds and their prepared concentrations

The work compounds Cinnamomum zeylanicum and sesame oils were obtained from local market in Jazan, Saudi Arabia. The following seven concentrations (15.37 µg/mL, 31.75 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL, and) of cinnamon oil were prepared in ethyl alcohol 70%. In addition, five concentrations of sesame oil were prepared, also in ethyl alcohol 70%; 3000, 95, 187, 375, 750, and 1500, µg/mL. These concentrations were dependent on our pilot work for the activity of each oil when used alone. The binary mixtures between cinnamon and sesame oils were done at a ratio of (1:3); one part cinnamon oil and three parts sesame oil as follows; (32 + 95), (62.5 + 185), (125 + 375), and (250 + 750), µg/mL.

2.2 GC–MS of the essential oils

The cinnamon and sesame oils were analyzed using GC–MS and TRACE GC Ultra Gas Chromatographs at an Educational Research Center, Nawah Scientific, in Egypt (https://nawah-scientific.com/) (THERMO Scientific Corp., USA).

2.3 Essential oil nanoemulsions preparation

Nanoemulsions were created using the Nerimela et al. [22] technique. Briefly, the essential oils were mixed with Tween 80 as a surfactant (1:3; one part oil to three parts T80) before being combined with water to achieve a concentration of 2.50%. Then, this was stirred with a magnetic stirrer for 10 min, at 500 rpm. An ultrasonicator (750 W, Branson Probe sonicator-Advanced model, 20 kHz) was used to sonicate the created macroemulsion for 5 min to create a nanoemulsion.

2.4 Characterization of nanoemulsions

A UV–visible spectrophotometer (UV-2600, Shimadz, Japan) set to 345 nm was used to analyze the produced nanoemulsions. Using a zeta sizer device (dynamic light scattering technique) (Nano-ZS90, Malvern, UK), the distribution of the droplet size (d, nm) and polydispersity index (PDI) of the produced nanoemulsion were measured. To lessen the impact of various scattering effects, all samples were diluted by 10% with deionized water.

2.5 Preparation of C. pipiens larvae

C. pipiens colony that was raised in a lab had its egg rafts sieved into water-filled plastic containers. In enamel dishes with 1 L of unchlorinated water and 0.15 g of 50:50 lactalbumin (brewer's yeast), the acquired larvae were put. Every other day, the water was changed, and every day, food was added. In aluminum cages (0.51 m3 in size) with screens, adult mosquitoes were housed and fed on 10% cotton wicks soaked in a sucrose solution. The bug female was fed blood by a restrained quail. The deposited egg rafts were collected in a 400 cc plastic container. The colony was maintained at 26 °C, 75% relative humidity, and a 16 L: 8 D photoperiod. The bioassays utilized the third and fourth larval instars [23].

2.6 Larvicidal bioassay

This experiment followed the guidelines established by the World Health Organization [24]. The 250 mL plastic cups used for this test were used. The working solution was created by adding an aliquot of one mL of these dilutions to 99 mL of distilled water after the essential oils had been dissolved in ethyl alcohol at the specified quantities. Twenty third-instar/fourth-instar C. pipiens larvae were placed in groups of five for each concentration in plastic cups. One mL of the solvent that had been dissolved was utilized in the negative control. To determine the average death rate, motionless larvae were recorded after 24 h and presumed dead [25].

2.7 Residual effect bioassay against larvae

Four concentrations (125, 250, 500, and 1000, µg/mL) were prepared for each form of cinnamon essential oil. These concentrations caused at least 75% mortality of larvae (concentrations were higher than the LC50 of each cinnamon formulation). Meanwhile, three concentrations of sesame oil were selected (750, 1500, and 3000, µg/mL). For the binary mixture of C-SO, only two concentrations were tested: 1000C + 3000SO, and 500C + 1500SO. These concentrations were applied to the larvae, with exchange the treated larvae (dead and live) from each concentration after 24 h and replaced by new ones. This exchange was done every 24 h up to 144 h. A double lethal dose of deltamethrin double was applied as a comparative positive control.

2.8 Statistical analysis

For each treatment, five replicates were performed, and mean and SE values were computed. ANOVA was used to analyze larval mortality, followed by Duncan's multiple range tests (p 0.05). The LC50 and LC90 values, together with their respective 95% confidence intervals, were calculated using probit analysis. SPSS for Windows (version 22.0) was used for all statistical analysis. The Synergistic Factor (SF) was calculated by dividing the LC50 value of the individual test insecticide with the corresponding LC50 value of the test insecticide + synergist mixture [26].

$${\text{Synergistic}}\;{\text{ratio }} = \frac{{{\text{LC}}_{{{5}0}}\, {\text{of}}\;{\text{insecticide}}\;{\text{alone}}}}{{{\text{LC}}_{{{5}0}} \;{\text{of}}\;\left( {{\text{insecticide}} + {\text{synergists}}} \right)}}$$

Synergistic ratios of less than one are described as antagonistic effect, ratios greater than one are described as synergistic, and the ratios equal to one are described as have no effect.

3 Results

3.1 GC–MS phytochemical composition of cinnamon essential oil

Analysis of the chemical composition of cinnamon essential oil revealed that the chief compotent is cinnamaldehyde, (E)- (63.42%), and cinnamaldehyde dimethyl acetal was (13.16%), 2-propenoic acid, 3 phenyl-methyl ester (6.09%), cinnamaldehyde à-hexyl- (11.75%), and traces of other constituents (Table 1).

Table 1 The phytochemical composition of cinnamon by GC–MS
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3.2 Characterization of cinnamon essential oil nanoemulsion

The hydrodynamic particle size showed a mean droplet size of around = 391.7 d, n, with a PDI = 0.979. This low PDI value indicates that the droplets had a homogeneous size distribution (Fig. 1 A, B).

Fig. 1
figure 1

A The wave length of cinnamon nanoemulsion, B Size of cinnamon nanoemulsion

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3.3 Larvicidal activity of cinnamon formulations

The LC50 of cinnamon nanoemulsion against Culex pipiens larvae was 33.8 µg/mL, While the LC50 of the the ordinary form was 85.3 µg/mL for. Similarly, the LC90 for the nanoemulsion form was 65.1 µg/mL compared to 179.00 µg/mL for the crude form (Table 2, 3). For sesame oil,, the LC50 of the oil itself was 1265 µg/mL, but this decreased to 159.00 µg/mL when mixed at a ratio of one part cinnamon oil to three parts sesame oil (Table 2, 4). The larvicidal activity of sesame oil was 100% at concentrations of 3000 µg/mL and 60% at concentrations of 1500 µg/mL and,. The larvicidal activity at lower concentrations was not significant (Table 4). The reference insecticide (1.7 ug/ml deltamethrin) showed 100% mortality (Table 2). The cinnamon sesame oil combination at a ratio of 1:3 exhibited a 100% mortality rate of larvae at all applied concentrations (Table 5). These results suggest that cinnamon and sesame oils have a synergistic larvicidal effect, increasing the potency of sesame oil by tenfold (synergistic factor SF), and the potency of cinnamon oil by twofold (synergistic factor SF), as shown in (Table 3).

Table 2 Larvicidal mortalities of cinnamon oil and its nanoemulsion against Culex pipiens larvae (N = 20 larvae)
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Table 3 LC50 and LC90 of cinnamon oil formulations of against Culex pipiens larvae
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Table 4 Larvicidal activity of sesame oil (SO) against Culex pipiens larvae
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Table 5 Larvicidal activity of SO + cinnamon at a rate of three parts SO to one part CIN (3:1) against Culex pipiens larvae
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3.4 Residual effect of different formulations of cinnamon essential oil

Higher concentrations of cinnamon essential oil resulted in increased residual effect. Specifically, the highest concentration, 4131 ug/mL, exhibited larvicidal effect that lasted up to 96 h (Table 6). Regarding the nanoemulsion form of the cinnamon oil, when used at a concentration of 1122 ug/mL, its efficacy remained stable for up to 120 h (Table 6, 7). Sesame oil showed the shortest period of residual effect (74% after 48 h at the highest concentration 3000 ug/mL which decreased to 27% after 72 h (Table 8). However, the residual effect of the mixture of cinnamon and sesame oil mixture was not significantly different from that of each oil singly (Table 9). AS for the positive control, deltamethrin (3.5 ug/L) demonstrated a prolonged residual effect lasting up to 144 h (Table 9).

Table 6 LC50 and LC99 of cinnamon oil formulations of residual effect against Culex pipiens larvae
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Table 7 Residual effect of cinnamon essential oil formulations of on survival of Culex pipiens larvae
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Table 8 Residual effect of SO on survival of Culex pipiens larvae
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Table 9 Residual effect of a cinnamon-sesame oil combination at a rate of one part cinnamon to three parts sesame oil on the survival of Culex pipiens larvae
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4 Discussion

The first line of defense against mosquitoes should be directed to control the larvae. Therefore, larvicidal control has the most significant impact on reducing mosquito populations. Extensive use of chemical insecticides is associated with insecticide resistance, environmental damage, and public health risks [5]. In this context, it is significant that cinnamon essential oil possesses an efficient insecticidal activity [27]. The analysis of the essential oil C. zeylanicum in this study revealed Cinnamaldehyde, (E) to be the predominant constituent (63.42%). Other studies have also identified cinnamaldehyde as the main component in cinnamon essential oil, albeit in different concentrations [28, 29]. Previous studies on the insecticidal activity of cinnamon/cinnamaldehyde have mainly focused on agricultural insects, demonstrating efficacy against mealworm (Alphitobius diaperinus) and Anopheles gambia [30], and the larvae and pupae of Culex quinquefasciatus [31], against Culex quinquefasciatus and Musca domestica [32], as well as adults, eggs and larval stages of Culex quinquefasciatus [33], and a potent antioxidant effect and activity against piroplasm in C. cassia [34,35,36]. Another study has demonstrated that another competent of cinnamon essential oil, eugenol (96.5%), exhibited a more potent larvicidal effect against Anopheles Gambia compared to clove oil [37].

The LC50 of the tested C. zeylanicum oil against Culex pipiens larvae was 85.3 ug/mL but this decreased to 33.8 ug/mL when the nanoemulsion form was used. Other studies have found that the LC50 and LC90 values for the activity of the essential oil of C. zeylanicum L. against the Cx. Tritaeniorhynchus and Anopheles subpictus mosquito species are 124, 225 ppm, and 71, 123 ppm, respectively [38]. Previous work has similarly found that the LC50 value (24 h) of cinnamon oil against C. pipiens larvae to be 71.87 mg/L [39], while the LC50 value for cinnamaldehyde against A. aegypti larvae is 36 ppm [40] and 13.45 ppm [41]. A dosage of 100 ug/ml cinnamaldehyde obtained from Cinnamomum ‏osmophloeum leaf essential oil has been shown to induce 100% larval mortality of Aedes albopictus [28]. Another work has shown that cinnamon oil is toxic to Culex pallens larvae with LC50 66 ug/ml and LC90 99 ug/ml [42]. In contrast, another study found only weak larvicidal activity of cinnamon oil, with an LC50 of 429.75 ug/ml [3]. Cinnamomum zeylanicum also has larvicidal activity against Aedes stephensi, Aede. Aegypti and Culex quinquefasciatus, with LD95 of 228.2, 276.9 and 277.4 ug/ml, respectively [43]. Our results are therefore consistent with the most previous studies.

Regarding the mechanism of the lethal effect of cinnamon essential oils on mosquito larvae, [33] observed that Culex quinquefasciatus larvae develop a wrinkled body and loss of their inner respiratory tube, which is associated with a lack of oxygen, irregular breathing, and damage to the nervous system [44]. Another study has explained that the lethality is caused by damage to the digestive tract of larvae [45].

Nanoparticles have been shown to improve the insecticidal efficacy of various compounds [46]. In the present study, the nanoemulsion form of cinnamon oil was approximately three times more effective at killing mosquito larvae than the ordinary form (the LC50 was 85.3 ug/mL for the ordinary form but just 28.30 ug/mL for the cinnamon nanoemulsion form). Similarly, a nanoemulsion of Cinnamomum zeylanicum oil was up to 32% more effective killing against Anopheles stephensi larvae than the ordinary form [47]. Furthermore, a nanostructured form of C. zeylanicum essential oil can be used to control meal worm (Alphitobius diaperinus) with the least toxicity to the environment and its fundamental arthropods [27]. Work in related areas has also shown that a nanoemulsion of phytochemical oil was 1.4–1.6 times more effective against Sitophilus oryzae (the rice weevil) than essential oil [48]. Moreover, a nanoemulsion made with Pterodon emarginatus Vogel oil produced 100% mortality in Aedes egypti at a dose of 250 ppm, with effectiveness lasting for up to 24 h [49]. In addition, the nanoemulsion of Vitex negundo essential oil revealed significant larval toxicity against Aedes aegypti, with LC50 values of 43.29 ppm, again lasting for up to 24 h [50]. Finally, a nanoemulsion of mint achieved higher larvicidal activity against C. pipiens and M. domestica than the regular oil [51].

In this study, the larvicidal power of crude sesame oil was found to be quite weak, with an LC50 of only 1265 ug/mL. However When combined with cinnamon essential oil, at a ratio of one part cinnamon to three parts sesame oil, the LC50 decreased to 159.00 ug/mL. Overall, the addition of cinnamon to seasame oil at the rate of 1:3 increased the potency of seasame oil by a synergistic factor of 10, and increased the activity of cinnamon by a synergistic factor of 2. These findings align with those obtained by [52], showing that a range of essential oils extracted from the leaves of S. indicum had high lethal efficacy against Aedes aegypti (349.88 mg/L), An. stephensi (338.27 mg/L) and Culex quinquefasciatus (304.84 mg/L). Assitionslly, an 8:2 mixture of clove oil: sesame oil has been found to have a synergistic effect on lethal activity against adult Callosobruchus maculatus [18]. Moreover sesame oil has shown a synergetic effect with clove oil on the cotton leaf-worm, Spodoptera littoralis, and with acetamiprid against the larvae of Trogoderma granarium [53, 54].

The residual effect of both forms of cinnamon oil increased with increasing concentration. The ordinary form of cinnamon essential oil maintained a residual effect in water for up to 72 h when applied at a concentration of 1000 ug/ml, while the nanoemulsion form continued to have an effect for up to 120 h at the same dose. Similarly, a nanoemulsion of Cinnamomum zeylanicum oil was able to kill 100% of Anopheles stephensi larvae for up to three days [48], a nanoemulsion of Protium heptaphyllum resin essential oil had a residual larvicidal effect for 72 h after application against Aedes aegypti larvae at a dose of 40 ug/ml [51], and Eucalyptus oil retained a residual effect against Aedes mosquitoes for up to eight days at a dose of 1000 ppm [53].

It has previously been recognized that nanoemulsion forms can be applied more easily than the ordinary forms of essential oils since they have better solubility, supporting dispersion in aqueous media, and also better stability, thus increasing the residual time [54]. Nanoemulsions also benefit from reduced oil particle size, low surface tension, and a greater surface area, all of which help them to disperse the active components of the essential oils across the target site, and permeate the target itself. This explains why nanoemulsion technology offers better biological activity compared to bulk/original essential oils [55]. Additionally, the nanoemulsion forms retain essential oils basic safety and environmentally friendly characteristics since they are biodegradable and have few side effects on non-target organisms, and the environment [56].

5 Conclusions

Cinnamon essential oil has significant larvicidal activity which improved when delivered either in a nanoemulsion form or in a mixture of one part cinnamon oil to three parts sesame oil. This is an important finding since sesame oil is cheaper than cinnamon oil. Moreover, the nanoemulsion form of the cinnamon essential oil was found to maintain a residual effect against C. pipiens in water for longer than the ordinary form or a sesame oil formulation which may have a role in controlling house mosquito in Jazan district, KSA. Further investigation of the safety and environmental impact of these formulations to ensure they meet regulatory standards and can be safely integrated into mosquito control programs.

Availability of data and materials

It will be made available on request.

Abbreviations

C. pipiens :

Culex pipiens

EO:

Essential oil

C:

Cinnamon

CNE:

Cinnamon nanoemulsion

SO:

Sesame oil

CSO:

Cinnamon + sesame oil

PDI:

Polydispersity index

LC50 :

Concentration killed 50% of treated larvae

LC90 :

Concentration killed 90% of treated larvae

SF:

Synergistic factor

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Acknowledgements

The authors extend their appreciation to Dr. Ahlam Gamal (PhD) for helping in mosquito colony stablishment.

Funding

We have not received any funding.

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Authors and Affiliations

  1. Health Informatics Department, College of Public Health and Tropical Medicine, Jazan University, Jazan, Saudi Arabia

    Hesham A. Mahran

  2. Hygiene, Zoonoses and Epidemiology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, 52611, Egypt

    Hesham A. Mahran

  3. Parasitology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, 62511, Egypt

    Shawky M. Aboelhadid

  4. Animal Health Institute, Beni-Suef, Egypt

    Khaled M. Hassan

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  1. Hesham A. Mahran
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  2. Shawky M. Aboelhadid
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  3. Khaled M. Hassan
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Contributions

HAM contributed to Conceptualization, investigation, funding, writing and review; SMA contributed to Conceptualization, investigation, methods, analysis, writing and review; KMH contributed to Investigation, data curation, data analysis, drafting writing.

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Correspondence to Hesham A. Mahran.

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Professor Shawky M. Aboelhadid is a co-author of this study and a Managing Editor of the journal. He was not involved in handling this manuscript during the submission and review processes. The rest of the authors have no conflict of interest to declare.

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Mahran, H.A., Aboelhadid, S.M. & Hassan, K.M. Synthesis and efficacy of cinnamon oil formulations and their sustainable release against common house mosquito larvae. Beni-Suef Univ J Basic Appl Sci 12, 118 (2023). https://doi.org/10.1186/s43088-023-00455-9

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