Nanoemulsion: A Green initiative for pest management

Brinjal or eggplant is a very important crop (vegetable) of sub-tropics and tropics of India. This plant is highly infested by, Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae). It is a polyphagous pest in nature, the adult and grub stages cause severe damage to the plants by feeding on its leaves, flowers and vegetables and create a heavy economic loss. Though the beetles could be controlled using synthetic pesticides, indiscriminate use of pesticide in the field causes problems such as pest resistance, environmental and health hazards etc. These pave way for the development of effective ecofriendly pest control measures. Plant based essential oils are used against these insect pests, as they constitute a rich source of bioactive components and reported to have many biological properties. Recently, essential oils were formulated as nanoemulsion and was developed for their effectiveness and eco-friendly nature. In this study, the oil in water (O/W) nanoemulsion of Mentha piperita were prepared by ultrasonication method at different concentrations and examined for the stability study. The stable nanoemulsion was characterized by DLS and was reported to consist with a mean droplet size of 10.84 nm, PDI was 0.1 and zeta potential was -45 mv which proved the good stability of the nanoemulsion. To assess the efficacy of the formulated nanoemulsion, and bulkemulsion, it was screened against the adult beetles, Henosepilachna vigintioctopunctata at different concentrations for 96 hours and the resulted LC50 value of nanoemulsion was 15.84 % which was found to be very effective than the LC50 value of bulkemulsion, which was 60.25 %. Hence from the results obtained, Mentha piperita nanoemulsion may be used as an organic pesticide because of its higher efficacy against the adult beetles, Henosepilachna vigintioctopunctata.


Introduction
Brinjal or eggplant (Solanum melongena Linn) is one of the most important vegetables crops grown all over India (Sarker et al., 2006) and it is well known for its nutritive value. Eggplant is highly infested by many insect-pests, but the major pest that attack the plant is Henosepilachna vigintioctopunctata (Coccinellidae: Coleoptera). It is the most destructive and polyphagous pest that leads to the heavy economic yield loss (Ali et al., 2017, Anam et al., 2006& Rahaman et al., 2008, Dhamdhare et al., 1995. Both, the grubs and the adult beetles causes damage to the plants and infestation begins just after the hatching of eggs (Murata et al., 1994). The grubs were feed on the lower epidermal layer of the leaves and the adult beetles feed on the upper surface by scrapping the leaves (Imura and Ninomiya, 1978) [1-5].The synthetic pesticides were used to manage the pests (Samanta et al., 1999;Das et al., 2002) but there is always a growing concern regarding the adverse effects, due to the indiscriminate use of the pesticides in the field, such as contamination of pesticides residues in the food, pest becomes resistance and environment pollutions (Ogendo et al., 2008& Kranthi et al., 2002. Recently, studies have reported the use of the plant essential oils, and their bioactive chemical constituents may be considered as an alternate to synthetic insecticides (Rajendran and Sriranjini, 2008), Since these essential oils consist of the different biological activity, such as antibacterial, antioxidant, insecticidal, etc. (Irshad et al., 2011 andKumar et al., 2012). Although, the essential oils consist of various properties, there are also certain limitations which are due to their volatile and hydrophobic nature. So, these problems may be overcome by recent trends, such as formulating essential oils into nanoemulsion, due to their transparency, solubility in water and also less amount of essential oil is required (Qian et al., 2012) [6-11]. An attempt was made to formulate the Mentha piperita essential oil as nanoemulsion. Because, the genus M. piperita is commonly known as peppermint, it is a cultivated natural hybrid of two species, Mentha aquatic L and Mentha spicata L. (Saller, 2004) and it is cultivated worldwide (Foster, 1996 Before the treatment, Insects were maintained at room temperature around 33 º C with 67 ± 1% relative humidity and the fresh leaves were provided to the beetles.

GC-MS analysis:
JEOL, GC MATE II (GC Model), quadruple double focusing detector. One microliter of the essential oil was injected in the split less mode of the injection port of the GC column. 220 °C for about 1 min then 10 °C min -1 was setup as an inlet temperature. The Helium gas act as a carrier gas at the constant flow rate of 1.0 mL/min. Total run time was about to 60 min. The interface temperature (GC to MS) was set to be at 250 °C. The MS was in a scam mode and the temperature was 250 °C. Ions are obtained by an electron ionization mode. Molecular ions (mass range) were monitored for the identification, which was set 50-600 m/z peak area denoted the relative percentage of constituents.

Characterization of nanoemulsion:
To check the stability of the formulations, it was expressed to different thermodynamic study such as centrifugation, heating-cooling cycle and freezethaw cycle. After passing the study, the stable emulsion was considered for characterization of the particle size. Dynamic light scattering (Model: Nanotrac Wave II; Make-Microtrac Inc, (USA) technique was used for the analysis of particle size distribution, polydispersity index (PDI) and zeta potential. So, prior to the measurements the emulsion was diluted using double distilled water (1:100) and equilibrated at 25°C for 1 min. the measurements have performed and the average droplet size was expressed as mean diameter and the surface charge of the droplets was determined by measuring the electrophoretic mobility at 25°C and the values of zeta potential were expressed in mV.

Bioassay:
The bioassay was carried out to evaluate the insecticidal activity of M. piperita essential oil nanoemulsion and bulkemulsion to check its effectiveness against the agricultural pest the adult beetles, H. vigintioctopunctata. Both the emulsions were screened against beetles, at different concentrations such as 10, 20, 30 & 40 % (v/v). Each treatment contains of 10 beetles and the fresh leaves (similar sizes) were chosen for the entire treatments. By leaf dipping method, the fresh leaves have been dipped into emulsions and allowed the leaves to air dried about a half an hour before the treatment. The entire treatment was carried out for about 24, 48, 72 & 96 hours respectively. So finally, for every 24 hours the leaves treated with the emulsions at different concentrations has been provided to the beetles of the treatment groups and the leaves treated with the acetone alone has been provided to the beetles of the control groups. Each test was triplicated and the mortality was recorded after every 24 hours of the treatments and the percentage mortality was calculated. [24][25][26][27][28].

Statistical analysis:
All the data has been subjected to the mean value and standard deviation. By using the probit analysis method, the LC₅₀ values were calculated and the significance difference between the treatment were analysed with one way ANOVA and multiple comparison between the variable were determine with Turkey's post Hoc test (P < 0.05) using the software SPSS, version 24.

Results & Discussion 3.1 GC-MS analysis
In the present study, the chemical constituents of M. piperita essential oil were identified using GC-MS technique. There are 15 components were identified and the major component was found to be menthol with highest percentage 16.47% among the other constituents (Table 1 & Fig 1). The identified major component is in good agreement with Hashimoto et al., (2016) and Malheiros et al., (2016). Gershenzon et al., (2000) reported that the essential oils mostly consist of monoterpenes. Prakash et al., (2015) reported that the essential oils and their bioactive components together is responsible for the biological activity, So the menthol present in essential oil may be responsible for the insecticidal activity against the insect pests.

Characterization of Nanoemulsion
In the present study, the formulated emulsions after passing the thermodynamic stability tests, the stable emulsion was characterized with the particle size 10.84 nm, PDI value as 0.1 and zeta potential as -45 mV (Table 2 & Fig 2). The droplet diameter less than 300 nm is considered to be as nanoemulsion ( ) and lesser the PDI value, the formulated nanoemulsion will consider to be more stable (Shinoda & Saito, 1969). The zeta potential value range above the -25 mV and +25 mV proves to be stable emulsion, while ranging within the values were consider to be unstable emulsion (Hertault et al., 2003). From the overall values, the formulated emulsion was found to be more stable and considered it as a nanoemulsion [32][33][34][35][36][37].  Fig 3).

Toxicity against H. vigintioctopunctata
From the above results, the highest mortality has observed in the nanoemulsion than the bulkemulsion, it is due to the small size of the particle already reported by many researchers reported the eucalyptus oil nanoemulsion ensures the higher effective against the C. quiquefasciatus larvae, than its bulkemulsion. Abouelkassem et al., (2015) has also reported that the jojoba oil nanoemulsion has showed a higher efficacy against the adult beetles, Sitophilus oryzae than its bulkemulsion. Therefore, the M. piperita nanoemulsion were found to be very effective against the adult beetle, H. vigintioctopunctata then the bulkemulsion and also in good agreement with the other researchers already reported. [37][38][39][40][41][42].

Conclusion
Hence, based on the results M.piperita nanoemulsion may be used as an alternative for the synthetic pesticides to control the agricultural pest H. vigintioctopunctata and also considered as safer to the non-target mammalian organisms, and also for environment sustainability.