Nanoemulsion of Myrtus communis essential oil and evaluation of its larvicidal activity against Anopheles stephensi

1 Introduction

Malaria remains a preventable cause of serious illness and death worldwide. The disease is caused by Plasmodium parasites, which are transmitted to other people through the bites of infected Anopheles mosquitoes (Ward 2008). Infants, children under 5, pregnant women, HIV / AIDS patients, migrant workers, and travelers are at higher risk for malaria (Osanloo et al., 2019a, 2019b, 2019c). Anopheles stephensi is a major vector of the disease. This species is the most abundant Anopheles species in Iran's malaria-prone areas (Osanloo et al., 2017a, 2017b, Firooziyan et al., 2021). According to the World Health Organization, until 2011, the distribution of A. stephensi was limited to certain countries in Southeast Asia and the Arabian Peninsula. Since then, this vector has expanded to Djibouti (2012), Ethiopia (2016), Sri Lanka (2017), and Sudan (2019). In the Horn of Africa, the vector appears to be expanding from neighboring countries (Djibouti) to neighboring countries (Osanloo et al., 2019a, 2019b, 2019c). Anopheles stephensi quickly adapts to the local environment. That is why it spreads to new lands (Osanloo et al., 2018). In addition, it resists several classes of insecticides and poses potential challenges to be controlled (Organization 2005, Fathian et al., 2015, Osanloo et al., 2019a, 2019b, 2019c). The danger of pesticides to ecosystems, human health, and other living organisms have become apparent, which has led to increased attention to herbal pesticides, especially plant essential oils, in recent years (Khanavi et al., 2013, Osanloo et al., 2019a, 2019b, 2019c).

The main problem of using essential oils in nature is their high volatility and consequent instability. To deal with this problem, nanoformulations should increase the stability and efficiency of the desired compound (Guan et al., 2010, Osanloo et al., 2017a, 2017b, Osanloo et al., 2018, Osanloo et al., 2019a, 2019b, 2019c, Firooziyan et al., 2021). Due to the small size of the droplets and the resulting transparency and long-term physical stability (without any apparent coagulation, precipitation, or biphasic), Nanoemulsions cause more adsorption and reduced material consumption (Naseema et al., 2021). The term nanocapsule with dimensions of 10 to 1000 nm is used (Hemingway et al., 2006). Nanoencapsulation of the active component prevents chemical reactions between the active substance and light, moisture, and oxygen. It also reduces side effects, prolongs the shelf- life, and controls the release of the active substance (Hemingway et al., 2006, Sell 2006, Bergeson 2010). For example, the controlled release of insecticides may provide several months of efficacy in vector control applications (Yadegarinia et al., 2006). Preparation and fabrication of plant-based nanoemulsions could be an interesting alternative to chemical pesticides for controlling mosquitoes. Nanoemulsions are also non-irritation to the skin, have minimal toxicity, and are not flammable (Messaoud et al., 2005, Gurpreet and Singh 2018).

Myrtus communis is a small shrub that normally grows to a height of 1 to 3 m. This plant has an anti-hair loss, antifungal, antibacterial, anti-inflammatory, and anti-dandruff properties (Tuberoso et al., 2006, Akin et al., 2010).

The essential oils of M. communis showed different insecticidal activity against Culex pipiens, Aedes albopictus, Pediculus humanis capitis, Mediterranean flour moth Ephestia kuehniella Zeller, the Indian meal moth Plodia interpunctella Hubner and the bean weevil Acanthoscelides obtectus Say (Traboulsi et al., 2002, Ayvaz et al., 2010, Sumbul et al., 2011). In addition, the repellent properties of this essential oil have been reported against A. stephensi (Tavassoli et al., 2011, Kayedi et al., 2014).

In this study, the larvicidal activity of M. communis essential oil (MEO) against A. stephensi was investigated and for the first time, a nanoformulation of M. communis essential oil (MNE) was prepared. The larvicidal properties of MNE were compared with MEO according to the guidelines of the World Health Organization (Organization 2005).

2 Materials and methods

3 Results

3.2

3.2 Determination of droplet size and morphology of MNE

Ten MNEs were prepared. From DLS results, a solution with d50 ∼ 200 nm, d90 ∼ 400 nm, and span < 1 was selected as the best nanoformulation (F4) for the next steps. Fig. 1 shows the DLS of the selected sample.

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Fig. 1

DLS results of MNE.

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4 Morphology of MNE

The TEM image of F4 is given in Fig. 2. The MNE droplets were well-formed, and the particles were almost spherical.

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Fig. 2

TEM image of the MNE particles.

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4.1

4.1 MEO larvicide properties

The larvicide effect started from 12.5 µg/ml and increased with increasing concentration and at 100 µg/ml all larvae were killed. Based on probit analysis, calculated LC₅₀ and LC₉₀ values for the essential oil were 26.1 (17.56–39.44) and 46.2 (38.62–61.18) µg/ml, respectively (Fig. 3).

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Fig. 3

Probit regression line of A. stephensi larvae exposed to different interval concentrations of MEO.

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4.2

4.2 Comparison of larvicidal properties MNE with MEO

To compare the larvicide properties of MNE and MEO against A. stephensi larvae, equal concentrations of MEO and MNE (F4) were used in the test. As shown in Fig. 4, after 24 h, MNE had 40% more efficacy compared with MEO. Based on the t-test, p < 0.001 was obtained.

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Fig. 4

Comparison of larvicidal effects of MNE and MEO against A. stephensi larvae after 24 h.

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The MNE showed better larvicidal properties in the 8-day test compared to its MEO and was able to kill all larvae in up to three days. After the third day, larval mortality decreased and reached 44%. MEO was able to control 100% of the larvae only for the first day, and the larval mortality rate decreased from 92% to 16% from the second day to the sixth day. MEO did not affect the larvae on the seventh and eighth days of the experiment (Fig. 5).

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Fig. 5

Comparison of larvicidal properties MNE and MEO against A. stephensi larvae in 8 days.

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5 Discussion

Chemical pesticides cause serious damage to the environment and non-target animals (Guan et al., 2010). In addition, frequent use of pesticides has led to pests resistance. This has reduced the number of pesticides suitable for use in vector control programs, especially in the control of malaria and Aedes-borne diseases (Hemingway et al., 2006, Sell 2006). Nanoformulations, by providing faster and more absorption in the target pest, can increase the efficacy of pesticides (Bergeson 2010, Gurpreet and Singh 2018). Nano-formulations of plant pesticides could be an interesting alternative in the fight against agricultural pests and vector-borne diseases.

In this study, the number of identified components (i.e. 107) in MEO was more than in previous studies (i.e. 16 to 70) (Vanhaelen and Vanhaelen-Fastré 1980, Chalchat et al., 1998, Asllani 2000, Messaoud et al., 2005, Tuberoso et al., 2006, Yadegarinia et al., 2006, Akin et al., 2010, Zomorodian et al., 2013, Rasooli et al., 2018). Comparison and study of ingredients of EO are very important since different numbers of biologically active substances affect the biological activity of EO (Koutsaviti et al., 2015). Various factors can affect the chemical composition of the studied EOs. They include geographical location, the plant parts studied, type and method of extraction and/ or analysis of the EOs, method of drying, storage conditions, fruit ripening stage, type of the plant studied (wild / lab), cultivar and genotype of the plants (Parra and Amo-Marco 1998). It is difficult to establish a relationship between the larvicidal efficiency and the components of the essential oil, as the interactions and synergistic effects between the components can affect the activity of the MEO (Felipe et al., 2008, Scotti et al., 2014). However, it is well known that multi-component formulations are less likely to slow resistance in vectors. This is the main advantage of using EOs as pesticides.

Although the effect of many substances is still unknown, what is certain is that the vector resistance to pesticides is more common to a formulation with an effective component rather than a multi-component formulation (Intirach et al., 2012, Worthington and Melander 2013, Araujo et al., 2016).

A few studies have been performed on the larvicidal properties of MEO against important health vectors. A comparable result was obtained by Koutsavitia et al. (2015). They collected various taxa of the M. communis from different parts of Greece and extracted essential oils. All samples of Greek essential oils were tested against Culex pipiens. They reported moderate to weak larvicide properties compared with our results (LC₉₅ and LC₅₀ were 160 and 76.6 mg / l, respectively). Such differences may be attributed to the different chemical compositions of EOs were prepared from plants, although there was not a significant relationship between the chemical composition of different taxa of this plant in this study and their larvicidal activity (Koutsaviti et al., 2015). In 2010, Conti and colleagues investigated the larvicide properties of essential oils against Aedes albopictus. The results were shown at a concentration of 300 µg/ml only 36.7% of larvae were killed (Conti et al., 2010) while our results indicated 100% mortality at 50 µg/ml.

Plants EOs have been categorized based on the LC₅₀ into six categories (Vatandoost et al. 2012). According to their suggestion, plants EOs with the LC₅₀ values of less than 1 consider as extremely-active, 1–5 very active, 5–50 active, 50–100 moderately active, 100–200 slightly active, and more than 200 non-active respectively. The larvicide properties of MEO are active in this classification and can compete with chemical larvicides, we used nanoemulsion to increase the stability of the EO in nature. From our findings, the MNE increased the larvicide properties against A. stephensi by ∼ 40% compared to the bulk EO (Osanloo et al., 2017a, 2017b). This is due to smaller particles which contribute to improved penetration into the larvae. Also, the residual effect test showed complete larvicide activity for 3 days for the MNE and 1 day for the MEO. A similar pattern of results has been obtained previously. Firooziyan et al. (2021) investigated the larvicide properties of cinnamon essential oil and nanoemulsion against A. stephensi larvae. The results were shown a 32% increase in the larvicide effects of nanoemulsion of cinnamon essential oil compared to cinnamon essential oil. Nanoemulsion of cinnamon essential oil had a high larvicide effect for up to 72 h, while after 24 h the residual effect of cinnamon essential oil decreased (Firooziyan et al., 2021). Osanloo et al. (2021) achieved similar results. Nanoemulsion of Artemisia dracunculus essential oil increased the larvicide properties of its essential oil against A. stephensi from two days to nine days (Osanloo et al., 2019a, 2019b, 2019c). A comparable result was obtained by Volpato et al. (2016). They investigated the effect of cinnamon essential oil and nanoemulsion against Alphitobius diaperinus. Nanoemulsion at a concentration of 5% caused 70% mortality of larvae after two days and had a threefold effect compared to treatment with essential oil (Volpato et al., 2016). Balasubramani et al. (2017) also showed increased larvicide activity of nanoformulation of Vitex negundo L.essential oil compared to its essential oil against Aedes aegypti (Balasubramani et al., 2017).

6 Conclusion

The study showed that nanoemulsion of essential oil may play an important role in the control of larvae. The nanoemulsion also showed increased stability for the essential oils the main challenge when using essential oils as larvicides.