Synthesis of natural nanopesticides with the origin of Eucalyptus globulus extract for pest control

ABSTRACT Continued use of chemical insecticides causes toxicity to non-target organisms, pest resistance and environmental pollution around the world. Nanocapsules of botanical insecticides are a suitable alternative for this target. The major constituents of Eucalyptus extract were determined by GC-MS: 1,8-cineole (70.94%) and 1,2-benzenedicarboxylic acid (6.08%). The Characterization of nanocapsule was investigated by dynamic light scattering, Zeta potential, scanning electron microscope, optical microscope and FTIR. The mean particle size distribution of nanocapsule is 380 nm and zeta potential of −26.36 ± 5.31 mV was obtained. It could be observed that nanocapsules have a spherical shape. To determine the insecticides of the nanocapsule with loads Eucalyptus extract was impregnated with concentration (0, 10, 15, 25, 35, 50 mg/ml) for 12, 24 and 48-h after exposure. The results indicated, that over time encapsulation exposure from 12 to 48 h, the highest time was comparatively more susceptible (LC50 = 14.93 mg/ml). GRAPHICAL ABSTRACT


Introduction
The green peach aphid, Myzus persicae is the most economically important agricultural crop pests in Iran. Myzus persicae causes damage to its host by direct feeding, the transmission of plant viruses and the production of honeydew (1)(2)(3)(4). This widespread use of synthetic pesticides for agricultural has resulted in the presence of their residues in rivers, lakes, sea, soils, groundwater, and even drinking water, proves the high risk of these chemical insecticides on human health, toxicity to nontarget organisms and pest resistance (5)(6)(7)(8)(9). The first case of chemical insecticide resistance in Myzus persicae was reported by Anthon in 1995 (10). Many researchers have indicated that chemical insecticides cause qualitative and quantitative changes in the soil microbiota (11,12). The use of plant materials as botanical pesticides for the protection of farm crops against insect attack has a long history, botanical pesticides such as Thymus vulgaris (13), Azadirachta indica (14), Rosmarinus officinalis (15), Artemisia absinthium (16), Piper nigrum (17), Artemisia vestita (18) Crysanthemum cinerariaefolium (19), tea leaves (20). The botanical pesticides are an excellent alternative to synthetic pesticides as a means to decrease negative health effects, and non-target organisms and the environment (21)(22)(23). Eucalyptus is known to contain bioactive compounds that indicated antifungal, antibacterial, antioxidative, fumigant and insecticides activities (24)(25)(26)(27)(28).
Encapsulation of plant extracts and essential oils suitable method to improve stability, persistence, and protect them from adverse environmental conditions such as undesirable effects of light and moisture (29). Encapsulation is a technology, which is specifically suitable to stabilize and control the release of bioactivity of natural extracts (30). Encapsulation technology has been applied to develop controlled-release types of pesticides for crop protection (31). Several encapsulation processes rely on the in situ polymerization technique and have been assayed briefly by different researchers (32). Nanopesticides consist of either very small particles of a pesticide active component (33). During the past few years, a few researchers published studies on natural pesticides with nano-encapsulated covers such as Moretti and et al, investigated the effect of microcapsule and emulsions of R. officinalis L and Thymus herbal-Barona Loisel on Lymantria dispar L. that''s one of the most serious pests cork oak forests. The result indicated that the encapsulation was a suitable method for protecting essential oils of very a different chemical ingredient (34).
There is, however, no report on the insecticidal activity of hydroalcoholic Eucalyptus globulus extract and also nano-encapsulation of E. globulus extract as a green synthesis on the Myzus persicae. This paper first time report on the manufacture of urea formaldehyde (UF) nanocapsules loaded by E. globulus extract prepared by in situ polymerization in an oil-in-water emulsion. Our study aimed at developing insecticides using the nano-encapsulated Eucalyptus extract that is economical and safe. The nanocapsules Eucalyptus extract was used as an insecticide carrier for the first time against Myzus persicae. As the new technology in nano-encapsulated Eucalyptus extracts through the controlled release of active ingredients overcome the restrictions of plant extract usage in laboratory condition. In the present study, the insecticidal activity of the nano-encapsulation of Eucalyptus extract and pure extract on Myzus persicae was investigated.

Insect tested
Myzus persicae adults collected from greenhouses and farm from Anzali port in the north of Iran. The Myzus persicae were held with the host plant in mesh glass cages in 50 × 60 × 60 cm under greenhouse conditions. Myzus persicae stock was maintained at 25 ± 1°C and humidity 64 ± 10%.

Preparation of the plant extract
The E. globulus was collected from the Anzali port in the north of Iran in August 2017. The collected plants were washed with tap water and finally rinsed with distilled water to remove impurities. The plants were dried at room temperature, and then the sample was crushed by an electric mixture to powder. The sample was kept in a closed glass. The powder of Eucalyptus provided after grinding was used for extraction. 10 g of the powder Eucalyptus was refluxed in a Soxhlet apparatus in 100 ml of ethanol and water (30 vol% alcohol and 70 vol% distilled water). After 6 h, the hydroalcoholic extract was filtered through Whatmann No.42 filter paper, and the filtrate was dried to evaporate the organic solvent and water by freeze-dried apparatus (Lyophilization), then the sample was kept in the refrigerator at 4°C.

Preparation of Eucalyptus extract nanocapsule formulation
. The preparation of the nanocapsule formulation of the Eucalyptus extract was performed by in situ polymerization technique using emulsion based on the oilin-water method (35 (36). Next, 2 (g/ml) of Eucalyptus extract was added slowly and after 30 min, the pH of the solution was adjusted by sulfuric acid 1 N, until the poly urea-formaldehyde shell covers the particles of the extract. The creamy viscous liquid was produced after 3 h. At the end of the reaction, the liquid mixture was cooled by the addition of distilled water. After one hour cooling, nanocapsule product appeared as a cream solid. Moisture evaporates from samples by oven-drying. Place samples in an oven at approximately 50°C for 24 h, and then samples dried. Finely, F4 was chosen for insecticides activity studies as optimum formulation due to lowest UF value and high agitation rate (rpm). Table 1 represents the composition of nanocapsules prepared at different processing parameters (37). The mechanism of urea-formaldehyde formation was shown by Siva and Sathiyanarayanan (38).

Characterization of nanocapsule
Nanocapsule morphology and size were determined SEM and OP. Dynamic light scattering technique has been applied to assign the mean size of nanocapsules (F4). The encapsulation yield was calculated from the equation (1). N is initial the weight (g) and C is weight of nanocapsule produced (g), (39) Zeta potential: Zeta potential of synthesized nancapsules was analyzed to determine the charge present on the nanocapsules (40

The release profile of encapsulated plant extract
The release profile of encapsulation plant extract was evaluated at 25°C and 65% relative humidity. 2.00 g of the nanocapsule, placed in a plastic Petri dish covered with a nylon net. After (1, 3, 7, 11, 15, 25 days) the amount of the residual content of the encapsulated plant extract was weight. Each experiment was replicated three times. The release profile of encapsulation plant extract was done by a method (41). The investigation of toxicity durability Eucalyptus extract and nanocapsule with loads Eucalyptus extract on Myzus persicae was investigated at 25°C and 64% relative humidity. 50 mg/ml of the nanocapsule and 60 mg/ ml of Eucalyptus extract, was prepared. Filter paper (Whatman No. 42) was impregnated with 1 ml of nanocapsule and Eucalyptus extract, the impregnated filter papers were then attached to the plastic Petri dish (8 cm diameter), twenty-five of Myzus persicae released into Petri dishes. In the same release period (2 and 30 days after preparation of nanocapsule (50 mg/ml) and pure Eucalyptus extract (60 mg/ml) in room temperature), the mortality of the test Myzus persicae after 48 h of exposure when treated with nanocapsule and pure Eucalyptus extract, nanocapsule with loads Eucalyptus extract compared to pure plant extract.

Detection of secondary metabolites in the plant extracts
The characterization of chemical compounds in the hydroalcoholic Eucalyptus globulus extract is shown in Table 2. The major chemical compounds in the hydroalcoholic extract were found (Table 2)

Fourier transform infrared (FTIR) Spectrophotometer
The FTIR spectrum of the pure Eucalyptus extract and nanocapsule (F4) recorded in the 4000-500 cm −1 range is indicated in Figure 1(a, b). The Figure 1(a) shows the   assigned to the C-X (Halogen; Vibration of halogen and out of plane) (53). Figure 1( (54). The peak at 578 cm −1 is assigned to C-X (Halogen; Vibration of halogen and out of plane). The peak at 911.94 cm −1 is assigned to -C-H Alkenes (vibration mode: out of plane). The peak at 2084.35 cm −1 corresponds to N=C.

Characterization of nanocapsules
Morphology of the nanocapsules (F1-F4) was determined by scanning electron microscope (SEM) and optical microscope of nanocapsule (F4: optimum formulation). SEM images of nanocapsule (F1-F4), Dynamic light scattering (Nanocapsule: F4) and optical microscope (OM) image (F4) was illustrated in Figures 2-6. It could be observed that nanocapsules have a spherical shape. The particle size distribution of nanocapsule (F4) is 380 nm (Figure 7). The mean zeta potential of synthesized nanocapsules was found to be −26.36 ± 5.31 mV which indicates that particles were relatively stable (40). The zeta potential diagram of nanocapsule was indicated in Figure 7. There are two spectrums of the nanometric particle (1, 2) in treatment which particles under 100 nanometers can be found and make nanocapsule form. The morphology results of nanoparticles loaded with garlic essential oil and nanoparticle size indicated that the nanoparticles had a spherical shape and 240 nm in the average size distribution (28). The

Release profile of nanocapsule
The pattern of plant extract release from the nanocapsule is shown in Figure 8. The amount of the residual content of the encapsulated plant extract after 1, 7, 14 and 21 days decreased from 100% to 96.5%. When exposed to room temperature and a moist environment, nanocapsule initially absorbed water. This absorption led to some modification in the wall materials (34). After 7 days of exposure, no further decrease in the sample weight was observed. Therefore, it can be concluded that encapsulation of the extract plant can significantly prevent a fast weight loss. Over time, the gradual release of the extracts from the nanocapsule was observed.

Determination of Eucalyptus extract insecticidal activity and Eucalyptus extract and UF nanocapsule containing Eucalyptus extract core on insect
In the present investigation, the insecticidal activity of Eucalyptus extract nanocapsule was evaluated against Myzus persicae. From the Table 3, it can be seen that the highest concentration (50 mg/ml) of the Eucalyptus extract encapsulation yielded 100% mortality of Myzus persicae at 48 h exposure. The mortality of Myzus persicae was increased with an increased solution concentration and exposure time of Eucalyptus extract nanocapsule. These results agree with researchers (29,41). The total mortality of all insects was achieved with the highest concentration and over time, presumably due to the slow and the persistent release of the active components from the nanocapsule (29). Probit analysis showed in Table 4, that over time encapsulation exposure from 12 to 48 h, the highest time was comparatively more susceptible (LC 50 = 14.93 mg/ml). Tukey''s HSD test showed the significant difference between the treatment (Sig <0.05) in (Table 4). Results show that concentration and time were all significant for the mortality of test pests after two days of exposure when treated with encapsulated Eucalyptus. The insecticidal activity of Eucalyptus extract at different concentrations is shown in Figure 9. From the Figure, insecticidal activity appears at 5 ppm and was increased with an increased in Eucalyptus extract to 60 mg/ml, which provides a perfect insecticidal activity for 48-h exposure. 60, 80 and 100 mg/ml plant extract gave 100% mortality and were significantly (F = 1630.350, df = 8, Sig = 0.000) at 48 h. LC 50 of the testing Eucalyptus extract was determined to be 16.95 mg/ml for 48-h exposure (Table 5). Insecticidal activities of nano-encapsulated Eucalyptus globulus extract with UF were not reported against insects till data as evidenced by researchers. The repellent and insecticidal effects of E. globulus essential oil with different nanostructures (nanoemulsion and nanocapsules) were tested on Haemotobia irritans flies and Musca domestica. After 24-h, a significant decrease in horn flies' population was observed using free and nano-encapsulated forms investigated. Eucalyptus   indicated 100% mortality of Culex tritaeniorhynchus rate within 12-h (56). Insecticidal activities of E. globulus extract were not studied against pests till data as evidenced by researchers, while the insecticide activities of E. globulus oil were investigated on insects. Abdel and Morsy, reported that essential oils from E. globulus showed toxicity against M. domestica (57). In a similar study, effects of E. globulus essential oils against Lasioderma serricorne F. and Rhyzopertha dominica F. were investigated by using the fumigation method. The mortality of Lasioderma serricorne F. and R. dominica was increased as the exposure time and the dose of the E. globulus oil increased. Obtained results suggest that toxicity properties of E. globulus oil can be proposed as alternatives to chemical pesticides for controlling R. dominica and L. serricorne (58). The summary of the literature about the natural insecticides with major compounds, including 1,8-cineole in Table 6 was presented (59)(60)(61)(62)(63). In the present study, the mortality of Myzus persicae was increased as the exposure time and the dose of the nanoencapsulated Eucalyptus globulus extract increased. The toxicity durability of Eucalyptus extract ( Figure 10) 100% mortality and for nanocapsule, 100% mortality for 2 days. The toxicity durability of Eucalyptus extracts 8% mortality and for nanocapsule, 72% mortality for 30 days, presumably due to the slow and the persistent release of the active components from the nanocapsule (29). The result was indicated, that the encapsulation is a suitable method for protecting plant extract of very a different chemical ingredient. Nowadays by using new technologies such as nano-encapsulated formulation can overcome the limitations of plant extract and essential oil (34). In this study, the plant extract of Eucalyptus was encapsulated by in-situ polymerization of oil/water. Fumigation assay was conducted to investigate the toxicity of the E. globulus against adults of Myzus persicae.  The effective fumigation assay of Eucalyptus globulus extract was basically due to the presence of the volatile constituents such as monoterpenes (1, (64,65). The mechanism of action of these compounds exerts their neurotoxic effects on insects involving multifarious mechanisms such as inhibition of nerve conduction enzyme acetylcholinesterase and octopamine synapses (66).

Conflict of interest
The authors disclose that there is no any conflict of interest in connection with submitted manuscripts. Mohammad Zaefizadeh is an assistant professor of agriculture Faculty of Biology, Ardabil branch, Islamic Azad University, Ardabil, Iran. He published more than 10 papers in international journals.

Disclosure statement
No potential conflict of interest was reported by the authors.