Preparation of nanoemulsion of Cinnamomum zeylanicum oil and evaluation of its larvicidal activity against a main malaria vector Anopheles stephensi

Abstract

Purpose

There is a growing need to use green and efficient larvicidal as alternatives for conventional chemicals in vector control programs. Nanotechnology has provided a promising approach for research and development of new larvicides. Larvicidal potential of a nanoemulsion of Cinnamomum zeylanicum essential oil reports against Anopheles stephensi.

Methods

The nanoemulsion of was formulated in various ratios comprising of C. zeylanicum oil, tween 80, span 20 and water by stirrer. It was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). All components of C. zeylanicum essential oil were identified by GC–MS analysis. The larvicidal potential of the oil and its nanoformulation were evaluated against larvae of An. stephensi. The stability and durability of nanoemulsion was observed over a period of time.

Results

Sixty one components in the oil were identified, cinnamaldehyde (56.803%) was the main component. The LC₉₀ and LC₅₀ values of C. zeylanicum essential oil were calculated as 49 ppm and 37 ppm, respectively. The nanoemulsion droplets were found spherical in shape. It was able to kill 100% of larvae in up to 3 days. It was stable after dilution and increased its larvicidal activity up to 32% compared with the essential oil.

Conclusions

A novel larvicide based on nanotechnology introduced. This experiment clearly showed increasing larvicidal activity and residual effect of the nanoformulation in comparison with the bulk essential oil. It could be concluded that this nanoemulsion may be considered as safe larvicide and should be subject of more research in this field.

Graphical abstract

Introduction

Vector-borne diseases (VBDs) are infection diseases caused by parasites, viruses or bacteria that are transmitted by vectors, such as human malaria which is transmitted by Anopheline mosquitoes. Malaria is still one of the major health problems worldwide. In 2019, World Health Organization reported approximately 229 million cases of malaria and approximately 409,000 deaths from malaria [1]. Vector control is the principal method for controlling many VBDs. Fighting against mosquito vectors in the larval stage is one of the important strategies endorsed by WHO. Besides, for some VBDs such as dengue fever, the only way to control the diseases is to control their vectors [2]. Epidemiological variables of malaria are associated with high genetic diversity of parasites, rapid incidence of vector insecticides and dispersal of vector species that make Anopheles vector control challenging. Anopheles stephensi is one of the major malaria vectors in the Middle East and South Asia. Recently An. stephensi has expanded its distribution to Djibouti, Ethiopia and Sudan in Africa and Sri Lanka in Asia. This is a major public health issue and WHO considers it as a significant threat to malaria control and elimination in Africa and southern Asia [3].

Synthetic chemical-based insecticides are being frequently applied to control vectors of malaria. The emergence of insecticide-resistance of An. stephensi is now becoming a key challenge in controlling the spread of this vector [4]. Insecticides can contaminate environment in different ways and pollute various sources such as soil and water. In order to prevent environmental pollution by chemical larvicides, other strategies to control vectors are being considered. Essential oils (EOs) as natural products are considered safe alternatives to synthetic pesticides as they are commonly safe for mammals and have short environmental persistence. However, low water solubility and low residual effect of EOs has provided rationale for development of efficient formulations such as nanoemulsions [5]. Nanoemulsions are mainly used in many industries today due to their special size, transparent or semi-transparent appearance, droplet size distribution, low viscosity and high stability against the phenomena of sedimentation, creaming, coalescence and clotting [6, 7]. In recent years, nanotechnology has accounted for a large share of global trade in various fields. Eliminating water pollution with the help of nanostructures is an environmentally friendly method [8]. Nanoemulsions as nanoactivators are of great importance in pharmaceutical, health, and cosmetic formulations. They are useful in controlling and disseminating the drug, releasing the active ingredients throughout the skin, targeting the drug in specific parts of the body, receiving vaccines, gene carriers, and intravenous actions, due to the precise goals in the implementation route. They are also suitable for drug delivery or DNA plasmids through the skin after dermal application. Due to the proper release of fragrances, they are also used in the formulation of alcohol-free perfumes. In the chemical industry, they are used as a polymerization reaction medium. Nanoemulsions with the ability to increase the solubility of water-insoluble pesticides are also used in the agrochemical industry [9–18]. A higher degree of delivery to the target of site, low preparation costs and lower ecological toxicity make nanoformulations as ideal formulation of EOs [5, 19].

In the field of vector control, nanotechnology providing promising larvicidal agents. So far, about 2000 plant species belonging to different families have been identified as having insecticidal properties, but essential oils and extracts have volatile components that limit their use in natural environments [20–22]. Similarly, different properties of extracts and EOs for insect control have been demonstrated in Iran [23–28]. Regrettably, the larvicidal power of extracts and EOs are generally lower than synthetic larvicides. Recently, by formulating them as nanomaterials, attempts have been made to reduce their volatility and increase their residual effects. Several studies have been conducted on extracts and EOs as an important class of natural larvicides [29–32], but there are few available articles on nanoemulsions as larvicides.

Cinnamomum zeylanicum (Family: Lauraceae), popularly known as cinnamon, is a common spice used in foods and pharmaceuticals since antiquity. EO of cinnamon bark and leaf is widely used in food flavors, cosmetics and pharmaceuticals [33], which have antibacterial, antifungal, antidiabetic and antioxidant properties [34]. The purpose of this study was to investigate the larvicides effect C. zeylanicum essential oil (CEO), prepare C. zeylanicum nanoemulsion (CNE) larvicides and evaluate the durability and stability of the larvicides properties of CNE as natural and eco-friendly product.

Materials and methods

Materials and equipment

The raw materials and equipment used in the study are shown in Table 1. CEO (100% purity) obtained from Green Plants of Life Co (Iran) with no addition of any other contents. EOs are usually degraded by oxidation, polymerization, hydrolysis and intermolecular reactions, these can be accelerated by the presence of heat, oxygen, moisture, or the presence of metals. To prevent these changes, the essential oil was stored in dark glass containers, away from sunlight at 4–8 °C.

Table 1.

Materials and equipment used in the study

Materials & Instruments	Producer	Country
Cinnamomum zeylanicum Oil	Green Plants of Life	Iran
Tween 80	Merck	Germany
Span 20	Merck	Germany
Ethanol	Merck	Germany
DLS	K-ONE.LTD	Korea
GC- MS	Agilent Tecnology	US
TEM	Zeiss	Germany
Centrifuge	Eppendrof	Germany
Magnetic Stirrer	DOMEL	Slovenia

Mosquito raring

Anopheles stephensi larvae were used in this study. They were reared in the insectary at 29 ± 1 °C (by electric heater) with relative humidity of 70 ± 5% (by steamer) under 12 h lightness/12 h darkness (by timer lamp). The adult and maintenance cages were 30 cm by 30 cm by 30 cm high, the floor was wooden and other sides were nylon mesh. The stock culture of adult An. stephensi was fed twice a week with artificial feeding on sheep blood. The defibrillated blood of the sheep was poured into a glass and Parafilm was placed on it and put upside down on the cage. To adjust the temperature, a container of hot water was placed on it. The egg rafts laid were then transferred to enamel larval trays. The larvae were fed with fish flakes.

Determining the larvicidal effect of CEO and CNE

Larvicidal bioassay were performed according to the WHO guideline [35]. Logarithmic concentrations were prepared from essential oil and nanoemulsion of essential oil by dissolving in ethanol. In all tests, 3rd or 4th early instar larvae were used that had been eaten the day before. During the test no food was given to the larvae. In the tests, water at room temperature, PH = 7 and chlorine-free was used. One ml of diluted essential oil or nanoemulsion essential oil was added to 249 ml of water and stirred, 25 healthy larvae were added to each container. The number of living and dead larvae was counted 24 h after the start of the assay.

Determining the durability of larvicidal CEO and CNE

In the long-time test, 1 ml of essential oil or nanoemulsion essential oil was added to 249 ml of water, then 25 live larvae were added to solution. After 24 h and observing the test results, without changing the solution, the larvae (both dead and live) were removed from the containers and 25 new live larvae were added to the containers. The larvae were exchanged for up to 8 days. The tests were performed in 16 repetitions at 4 different replicates. In each replicate 2 control groups were considered that ethanol added to those with similar mentioned manner.

Statistical analysis

Mortality rate data of An. stephensi larvae were subjected to logarithmic concentrations of CEO by probit regression analysis. Lethal concentrations of 50% and 90% (LC₅₀ and LC₉₀) were calculated using probit analysis [36, 37], the regression line was plotted using Excel 2007 software.

If the control group mortality was less than 5%, the data from the bioassay tests were considered correct, when the control mortality was between 5% to 20%, it was corrected using the Abbott formula [36]. If the larvae became pupae or the larvae mortality were more than 20% in the control group, the test was repeated.

Preparation of nanoemulsion

In this study, a spontaneous method was used to prepare nanoemulsion. First, surfactant (Tween 80) and co-surfactant (Span 20) were mixed thoroughly. The essential oil was then added to obtain final concentration of 90% lethal concentration and stirred for 10 min at 600 rpm. Afterwards, water was added dropwise and stirred at 600 rpm for 38 min. Different concentrations of surfactant and co-surfactant at constant essential oil concentration were prepared and stored in a dark place at room temperature for 24 h. Dispersions were visually checked for any sign of phase separation, precipitation or creaming. Dynamic light scattering (DLS), K-ONE. LTD, (Korea) was used to determine the particle size (PS) of the prepared nanoformulations. Transmission electron microscopy (TEM) was used to confirm the PS and to investigate the morphology of the particles.

Analysis of essential oil by gas chromatography–mass spectrometry (GC-MS)

Components of CEO were identified by GC–MS analysis. The GC device isolates volatile compounds, and the mass spectrometer helps identify each of the separated components based on their mass properties with high accuracy. CEO was diluted using hexane and after finding the most suitable thermal programming of the column, it was injected into the apparatus and mass spectrums and chromatograms were obtained. GC–MS was performed on HP (Agilent Technology) 7890A Network GC System connected with a 5975C VL MSD with Triple-Axis Detector. CEO was analyzed using HP-5MS Fused silica column (Length: 30 m, I.D.: 0.250 mm& Film thickness: 25 μm). The GC–MS settings were programmed as follows; initial oven temperature was held at 40 °C for 1 min, rising to 250 °C at 3 °C/min. The injector temperature was maintained at 270 °C. Detector temperature was at 230 °C. Carrier gas used was helium (He 99.999%) at a flow rate of 1 mL/min. The constituents of the essential oil were analyzed using retention indices mass spectra of the compounds and compared with the standard mass spectra available in the device library and authoritative references. 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) [38].

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

Results

Larvicidal bioassay of CEO

The larvicide results of 5 different concentrations of CEO are shown in Fig. 1. Sample containing 128 ppm CEO was found to be the most toxic with 100% larval mortality. There was no mortality in the control groups. In regression line a positive correlation was observed between CEO concentrations and the probit mortality (Fig. 2). The LC₉₀ and LC₅₀ values of CEO against An. stephensi larvae were calculated as 49 ppm and 37 ppm, respectively.

Fig. 1.

Larvicidal activity of CEO against An. stephensi

Fig. 2.

Probit regression line of An. stephensi larvae exposed to different interval concentrations of CEO

Yields and chemical constituents of CEO

The yield of CEO was 0.5% (v/w) based on fresh weight. CEO was yellowish with a distinct sharp odor. Sixty one components were determined by GC–MS analysis, with five major components of CEO including Cinnamaldehyde (56.803%), Linalool (6.310%), dl-Limonene (6.237%), trans-Caryophyllene (4.697%) and Eugenol (3.895%) as listed in Table 2.

Table 2.

Chemical composition of the CEO

NO.	Retention time (RT) (min)	Compound	Peak area	%	Quality	Mol Weight (amu)
1	25.606	Cinnamaldehyde	1,511,649,639	56.803	97	132.058
2	17.157	Linalool	167,945,919	6.311	97	154.136
3	13.664	dl-Limonene	165,991,974	6.237	99	136.125
4	31.268	trans-Caryophyllene	125,002,534	4.697	99	204.188
5	28.889	Eugenol	103,671,489	3.896	98	164.084
6	32.496	trans-Cinnamyl acetate	98,266,329	3.693	97	176.084
7	44.469	Benzyl benzoate	81,272,658	3.054	97	212.084
8	13.448	Carvacrol	72,963,965	2.742	97	134.11
9	26.77	Cinnamyl alcohol	49,436,677	1.858	98	134.073
10	14.199	Benzenemethanol	40,704,915	1.530	96	108.058
11	32.617	alpha-Humulene	19,556,247	0.735	99	204.188
12	11.228	beta-Pinene	18,802,634	0.707	97	136.125
13	21.267	alpha-Terpineol	16,495,180	0.620	91	154.136
14	9.427	alpha-Pinene	16,474,543	0.619	96	136.125
15	13.741	1,8-Cineole	15,409,268	0.579	99	154.136
16	34.328	Zingiberene	11,565,807	0.435	96	204.188
17	19.473	l-Menthone	10,517,354	0.395	98	154.136
18	22.641	trans-Cinnamaldehyde	9,721,905	0.365	97	132.058
19	10.025	Camphene	9,579,001	0.360	98	136.125
20	37.757	Caryophyllene oxide	9,123,643	0.343	91	220.183
21	19.651	Isoborneol	7,792,915	0.293	97	154.136
22	33.819	Benzene	5,397,789	0.203	99	202.172
23	15.013	gamma-Terpinene	5,391,389	0.203	96	136.125
24	26.013	trans-Cinnamaldehyde	5,112,899	0.192	96	132.058
25	25.854	trans-Cinnamaldehyde	5,110,766	0.192	96	132.058
26	26.312	Thymol	4,587,550	0.172	86	150.104
27	19.008	3-Benzylidenecamphor	4,545,271	0.171	98	152.12
28	34.843	beta-Bisabolene	4,451,083	0.167	97	204.188
29	18.614	Dihydrolinalool	4,391,505	0.165	86	156.151
30	35.448	beta-Sesquiphellandrene	4,340,898	0.163	98	204.188
31	21.585	Isoterpinolene	4,267,300	0.160	94	136.125
32	18.748	trans-Limonene oxide	3,764,993	0.141	91	152.12
33	11.947	beta-Myrcene	3,592,467	0.135	96	136.125
34	18.531	Limonene oxide	3,503,639	0.132	96	152.12
35	27.432	2-Propenal, 2-methyl-3-phenyl-	3,379,580	0.127	70	146.073
36	20.421	l-Menthol	3,283,274	0.123	91	156.151
37	10.706	Benzaldehyde	3,241,273	0.122	94	106.042
38	44.291	Cedryl acetate	2,772,151	0.104	91	264.209
39	11.126	Sabinene	2,627,279	0.099	96	136.125
40	29.378	alpha-Copaene	2,552,346	0.096	99	204.188
41	23.684	Kurarinone	2,344,028	0.088	96	150.104
42	20.096	1-Borneol	2,319,219	0.087	86	154.136
43	12.481	l-Phellandrene	2,120,629	0.080	94	136.125
44	40.327	Benzyl ether	1,645,951	0.062	83	198.104
45	21.451	trans-Caryophyllene	1,526,557	0.057	64	122.11
46	19.963	Menthone	1,333,607	0.050	96	154.136
47	20.599	Terpinen-4-ol	1,223,776	0.046	96	154.136
48	8.937	Tricyclene	1,001,303	0.038	96	136.125
49	31.567	Aldrichimica acta	1,001,002	0.038	38	120.094
50	13.041	alpha-Terpinene	984,871	0.037	98	136.125
51	11.851	5-Hepten-2-one, 6-methyl-	918,811	0.035	95	126.104
52	30.683	trans-Caryophyllene	817,896	0.031	50	204.188
53	23.449	Pulegone	777,614	0.029	97	152.12
54	19.123	Limonene oxide	754,831	0.028	53	154.136
55	52.033	alpha-Thujene	750,049	0.028	50	136.125
56	55.793	1,5-Bis[dimethyl(chloromethyl)silyloxy]pentane	691,891	0.026	37	316.085
57	28.259	alpha-Cubebene	612,257	0.023	86	204.188
58	42.522	Cyclopropa	570,689	0.021	64	204.151
59	17.991	cis-Verbenol	565,783	0.021	52	152.12
60	9.173	Bicyclo	533,073	0.020	94	136.125
61	41.937	Nonadecane	468,792	0.018	91	268.313

Determination of droplet size and morphology of CNE

Based on previous studies, of the 10 nanoformulations made (Table 3) and determine the particle size (PS) of the prepared nanoformulations with using DLS device. The formulation with d50 of less than 200 nm, d90 of less than 400 nm and span of less than 1 was the best nanoformulation (F6 of CNE) for the next steps. Figure 3 illustrates DLS of selected samples.

Table 3.

The amount of ingredients used in making nanoemulsion of bitter C. zeylanicum essential oil and choosing the best nanoformulation based on the results of DLS (d50: The size at which 50% of the particles are smaller, d10:The size at which 10% of the particles are smaller, d90:The size at which 90% of the particles are smaller and span=d90 − d10/d50

Formulations	C. zeylanicum essential oil	TW80 (μl)	Span (μl)	Water (μl)	d50 (μl)	d10 (μl)	d90 (μl)	Span (μl)
F1	100	100	50	4750	9.89	4.12	30	2.616
F2	100	150	50	4700	2.58	1.36	8.46	2.75
F3	100	200	50	4650	24	5.35	45.5	1.67
F4	100	250	50	4600	5.18	1.56	12.2	2.05
F5	100	300	50	4550	6.16	1.97	20.2	2.95
F6	100	350	50	4500	220	143	359	0.96
F7	100	400	50	4450	diphasic	diphasic	diphasic	diphasic
F8	100	450	50	4400	diphasic	diphasic	diphasic	diphasic
F9	100	500	50	4350	diphasic	diphasic	diphasic	diphasic
F10	100	550	50	4300	diphasic	diphasic	diphasic	diphasic

Fig. 3.

DLS results of nanoemulsion containing C. zeylanicum essential oil

F6 formulation of CNE, which was selected as the best nanoformulation based on particle size results, was prepared using transition electron microscopy (TEM) to image the morphology of their particles. The results showed that CNE was made correctly (Fig. 4).

Fig. 4.

Transition electron microscopy (TEM) image of the nanoemulsion particles

Nanoemulsion physical stability

The nanoemulsion was stored at refrigerator temperature (4 °C) and at room temperature for 4 months. No phase separation or creaming was observed. No change was observed even after centrifugation.

Larvicidal effects of CNE and CEO

Figure 5 indicates the residual effect of CNE and CEO for 8 consecutive days. The nanoemulsion (CNE) was able to kill 100% of the larvae in the first three days of experiments. After the third day, the larval mortality decreased gradually, from 92% in fourth day, 80% in fifth day, 76% in sixth day and 52% in seventh day to finally 32% on the eighth day, respectively. The bulk of essential oil was able to control 100% of the larvae only for one day, the larval mortality decreased from 96% to 4% from the second day to the eighth day, respectively. Even at the end of the experiment the larval mortality due to CNE was eighth was 8-fold more than CEO. Although the same concentration of CEO and CNE in 24 h test were used, the larvicidal activity of CNE was stable after dilution and increased its larvicidal activity. Larvicidal effects of CEO was 60%, while it increased to 92% by CNE (Fig. 6).

Fig. 5.

Comparison of larvicidal activity of CEO vs. CNE in the long time (8 days)

Fig. 6.

Comparison of larvicidal activity of CEO vs. CNE in the short time (24 h)

Discussion

Nowadays, natural products are being considered for number of applications [39]. They provide green and efficient alternatives for vector control, having no harm to the nature. However, these substances are generally volatile and their effect is short lasting, therefore, their original forms should be formulated to be used as mosquito larvicides in the environment [28]. On the other hand, these products are not often water soluble, being usually much more soluble a second reason necessitating formulation of herbal extracts lese essential oils. This is made clear that nanotechnology can improve water solubility of these substances and increase their efficacy as larvicides [40].

EOs are complex mixtures that may contain over 300 different compounds [41]. They have a very high variability of their composition. The number of each compound of CEO in earlier studies were varied from 9 to 38 compounds [42–47], while we were able to identify 61 compounds of this essential oil, probably due to timely GC analysis used in this study.

The LC₉₀ and LC₅₀ values of CEO against An. stephensi were reported as 198 ppm and 55 ppm, respectively in pervious study [48] while the result of the present study is better than a result of previous report showing 49 ppm and 37 ppm, respectively. According to proposed categories of larvicidal activity of plant EOs against mosquito larvae, CEO lies in the third category as active plant [25].

To increase the stability of CEO in nature we used low temperature emulsification method for making the nanoemulsion. Nanoemulsions with a particle size of less than 20 nm have been reported to have better lactation power due to their improved penetration into the larval body [49, 50]. The larvicidal activity of the nanoemulsion is assumed due to the presence of cinnamaldehyde, which is the major component of the oil by GC–MS analysis. Although, the connections and interactions between all components of CEO need to be considered. This is the first study on nanoemulsion formulation using CEO as larvicide with span of less than 1 has been reported against An. stephensi. The results of this experiment clearly showed 32% increase in larvicidal effects of CNE in comparison with the bulk essential oil, which is probably due to droplet size distribution. CNE had a high residual effect for up to 72 h while it was reduced in bulk of essential oil after 24 h. A similar pattern of results was obtained by Osanloo et al. 2019, showing increased effect of their nanoformulation against An. stephensi from two to nine days [51]. Similarly Volpato et al. (2016) investigated the effect of essential oil and nanoemulsion of C. zeylanicum on mealworm (Alphitobius diaperinus). The nanoemulsion at concentration of 5% caused 70% mortality of the larvae after two days and had a three-fold more pronounced effect when compared to treatment with unemulsified essential oil within 3 days [52]. This is consistent with result was obtained by Balasubramani et al. (2017), their experiment showed increase in larvicidal activity of nanoformulation Vitexnegundo L. in comparison of bulk essential oil on Ae. aegypti [53]. A similar conclusion was reached by Sundararajan et al., they found that the nanoemulsion of Ocimum basilicum EO was more effective that the bulk essential oil on larvae of Cx. quinquefasciatus [54]. These findings are in agreement with the result of the present study, in which, there are worthy larvicidal and residual effects of prepared CNE against main malaria vector An. stephensi.We performed TEM as we have easier access to that instead of HRTEM. Although, it is appear that the data generated by the TEM is good enough for our study, it is recommended to use HRTEM in the future studies.

Conclusion

Our nanoemulsion larvicide based on C. zeylanicum essential oil was effective against An. stephensi. The result obtained successfully demonstrates the larvicidal activity, residual effect, and stability of C. zeylanicum nanoemulsion. The nanomodification significantly improves effectiveness of CNE as larvicide compared with bulk of the oil. EOs are rapidly volatile and low residual activity while, the nanomodification meaningfully increases the residual effect of CNE for up to 72 h. The residual efficacy of CNE shows this formulation is a good candidate for eco-friendly and safe larvicide and should be subject of more research in this field. Further research on natural products, especially essential oils to find the best candidate as larvicides is recommended. Future studies should focus on increasing residual efficacy of CNE through improvements in formulation technology. Moreover, conducting research on mode of action of such new formulations would help to better understanding on the biological effects and performance of nanoemulsion in the field of vector control. This approach may provide a solution to insecticide resistance problem of controlling vectors with no or minimal harm to environment.

Declarations

Conflict of interest

The authors declare no conflict of interest.