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Evaluation of critical parameters for preparation of stable clove oil nanoemulsion
⁎Corresponding author. Tel.: +98 11 3233 4204; fax: +98 11 3231 0975. najafpour@nit.ac.ir (Ghasem Najafpour Darzi)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In this work, the effects of various factors, including the ultrasonic duty cycle and intensity of ultrasonic irradiation, ultrasonication and clove oil content in production of clove oil nanoemulsion were investigated. In preparation of nanoemulsion Tween® 80/Span® 80 as nonionic surfactants via ultrasonic emulsification method was used. The average droplets size of clove oil nanoemulsion decreased with an increase in duty cycle; whereas pulsed ultrasound with proper intervals was more efficient than continuous ultrasonication. In order to replace the use of organic solvent and increase the dispersity of active ingredient, suitable emulsifiers were used. The nanopesticides made by ultrasound at optimum formula conditions were defined at ultrasonication time of 300 s, surfactants concentration of 5 wt%, hydrophilic–lipophilic balance number of 9, duty cycle 0.75% and ultrasonic intensity 208 W/cm2. The stability of droplets size of nanoemulsions for duration of 6 months was evaluated. Stable nanoemulsion clove oil with 10 wt% of clove oil content was formulated at optimum conditions with average droplets size around 43 nm at the beginning and after 6 months nanoemulsions re-sized and it was around 100 nm. A stable nanoemulsion of clove oil in water with suitable droplets size as a nanopesticide was prepared.
Keywords
Clove oil
Nanoemulsion
Nonionic surfactants
Oil in water emulsion
Ultrasonication
1 Introduction
The world’s escalating population has recently exceeded seven billions. In 2050, in order to feed extra population, the food demand in developing world is expected to increase by 50–100% (Alexandratos and Bruinsma, 2012). To boost agricultural productivity, integrated pest management (IPM) has implemented the application of effective pesticides for the prevention of crop yield losses. Chemical methods are still crucial in view of the fact that they convey significant, essential control of weeds, pests and plant diseases in most economical approach. Nevertheless, the extensive and intensive use of synthetic pesticides has globally accelerated the evolution of herbicide-resistant weeds in an exponential rate (Baldos and Hertel, 2014; Bazoche et al., 2013). The worldwide damage caused by pesticides is projected to be $100 billion per year (Alston and Pardey, 2014). The reasons are categorized into two groups: (1) high toxicity and non-biodegradability of pesticides and (2) lack of exact scientific formulations, i.e. only a few percentages of pesticides has been effectively used; many of them either been washed out or penetrated into soil, rivers (Balbus et al., 2013; Dorne and Fink-Gremmels, 2013; Hanjra et al., 2012; Moschet et al., 2014; Stone et al., 2014). Hence, it is necessary to search for safe, highly selective and biodegradable pesticides to solve the problem for long term toxicity to mammal or one must study an environmental friendly formulation of pesticides. In addition, one has to develop new techniques that should be employed to reduce organic load of pesticide, while maintaining high crop yields (Cantrell et al., 2012).
Eugenol (4 allyl-2-methoxy phenol; C10H12O2) is a major constituent of clove essential oil; that is an organic phenol compound. It has specification of antipyretic, analgesic, anti-inflammatory, and anesthetic effects. Based on latest conducted research, clove oil (eugenol) also has antimicrobial (Manganyi et al., 2015), antioxidant (Avila Farias et al., 2014; Gülçin et al., 2012), antifungal (Pinto et al., 2009), insect repellent (Shapiro, 2012), and anticancer (Dwivedi et al., 2011) activities. Minimizing eugenol side effects, with low toxicity, and non-metabolized residue, eugenol has wide application in pharmaceuticals (Kong et al., 2014), cosmetics (Prashar et al., 2006), dentistry (Xu et al., 2013), food (Hyldgaard et al., 2012; Vrinda Menon and Garg, 2001), agriculture (Isman, 2006) and pesticides (Isman, 2000; Jiang et al., 2012). The anti-insect activity of eugenol strictly depends on the organic structure of its phenolic ring with specific hydroxyl functional group. In grain treatment, Huang and his coworkers (Huang et al., 2002) demonstrated that different doses of eugenol were required to exterminate Tribolium castaneum and Sitophilus zeamais. Pessoa and his team (Pessoa et al., 2002) studied the inhibition of eugenol on small ruminants of Haemonchus contortus by ovum hatching test and obtained the highest inhibition rate when concentration was about 0.5%. Machado and his group (Machado et al., 2011) studied the effect of eugenol on the growth, activity, adherence, and ultra-structure of Giardia lamblia. Their results showed that eugenol inhibits the adherence of Trophosome after being monitored for 3 h and does not cause cytolysis. They also found that eugenol affected on cell shape and caused the coagulation of cytoplasm and autophagy in the cells. These investigations demonstrated that eugenol can be used as a drug to prevent Giardiasis and Verminosis.
An emulsion of clove oil is a kinetically stable system which is obtained by the dispersion of one liquid (dispersant) into another phase; where each liquid is immiscible or poorly miscible in the other, e.g. oil and water (Purwanti et al., 2015). Based on literature survey, the suitable droplet sizes for nanoemulsions are in the range of 20–200 nm (Shah et al., 2010). Due to small droplets size, nanoemulsions appeared to be transparent or translucent (Solans et al., 2005). The biggest difference between nanoemulsion and emulsion is in the size of the water particles. When the size of the oil particles become small, the stability of the emulsion significantly improved. To enhance the kinetic stability of such a system, surfactants usually add to oil–water mixture. A surfactant is an amphiphilic molecule that has a hydrophilic head group (polar region), which has a high affinity for water, and a lipophilic tail group (nonpolar region), which has a high affinity for oil (Anton and Vandamme, 2011). In addition, nanoemulsion of oil in water is not easily separated; new approaches like application of certain charges in an electric field may require (Hosseini and Shahavi, 2012; Hosseini et al., 2012).
Emulsion technology is generally applied for the encapsulation of bioactive compounds in aqueous solutions through the production of nanoemulsions. The high kinetic stability of nanoemulsions is a real benefit for encapsulation purposes and plays a critical role in retention of surface oil content of the product (Augustin and Hemar, 2009). Nanoemulsions, being non-equilibrium systems, cannot be formed spontaneously and consequently it needs energy input, generally from mechanical devices or from the chemical potential of the components. Therefore, nanoemulsion formation is generally achieved using high-energy emulsification methods such as high shear stirring, high-speed or high-pressure homogenizers, ultrasonicator, and microfluidizer. These methods supply the available energy in the shortest time and possess the most homogeneous flow to produce the smallest droplet sizes (Nazir et al., 2010; Sharif et al., 2012; Solans et al., 2005).
As it has been reported that clove oil was successfully used as pesticide. In 1998, clove oil (CAS # 8000-34-8) as pesticide was registered under United States Environmental Protection Agency (USEPA). Also, clove oil is classified as minimum risk pesticides and is not subject to federal registration requirements because of their active and inert ingredients are evidently safe for human use (USEPA, 2011). Furthermore, eugenol, as an active ingredient of clove oil has a broad spectrum insecticide (EcoPCO® D) sold by EcoSMART Technologies (Alpharetta, GA, USA) (Wilson and Isman, 2006).
Generally, use of oil in water nanoemulsions as a nanopesticide has great potential for the replacement of the traditional emulsified oil. Nanoemulsion is used due to highly stable product for long duration. The main reasons to use oil in water as nanoemulsion are reducing the use of organic solvent and increasing the dispersity, wettability and penetration properties of the droplets. Other advantages of using pesticide oil in water nanoemulsions are for improvement of the biological efficacy and reducing the dosage of pesticides.
In our testing trial, use of several pests such as Sitophilus granaries, Oryzaephylus surinamensis, and Sitotroga cerealella was initially examined with effective pesticides using nanoscale clove oil at several concentrations. The experiments were quite successful and all the pests were defeated. The exact experiment was conducted on pile of grain for long duration; as there was no pest reported on treated wheat.
Surfactants are belonging to a group of substances that meet certain characteristics such as good surface activities and available to form condensed interfacial film. For preparation of nanoemulsion use of surfactants is essential for stable droplet size. Tween® 80 and Span® 80 are commonly used as safe surfactants because of their high degree of compatibility with other ingredients and low toxicity, Span® 80 as a viscous, lipophilic, emulsifying liquid agent. Tween® 80, a hydrophilic in nature is a derivative of Span® 80. Tween® 80 is more soluble in water than in oil. For having stable nanoemulsion one has to balance the hydrophilic and lipophic properties. Therefore mixture of Span® 80 and Tween® 80 was used. These nonionic surfactants as uncharged molecules are also known as safe and biocompatible products; they are not affected by any pH changes of the mixture (Lv et al., 2014; Mahdi et al., 2011; Sagiri et al., 2012).
Clove oil was selected as a pesticide for the preparation of nanoemulsion. In this work, nanoemulsion of clove oil in water was formulated using nonionic surfactants such as Tween® 80/Span® 80 by ultrasonic emulsification method. Effects of duty cycle of ultrasonic exposure, ultrasonic intensity, sonication time and clove oil concentration in droplets size of nanoemulsion were investigated.
2 Materials and methods
2.1 Materials
Clove oil (CAS #8000-34-8) was purchased from Sigma–Aldrich (St. Louis, MO, USA); Polyethylene glycol sorbitan monooleate (Tween® 80) synthetic grade and sorbitan monooleate (Span® 80) also synthetic grade were purchased from Merck Millipore (Darmstadt, Germany). Water used in all the experiments was purified with a Milli-Q system consists of filtered through 0.2 μm filters (Millipore Co., Bedford, MA, USA).
2.2 Ultrasonication
In this study, emulsification by sonication was prepared using an ultrasonic processor (model UP400S, powermaximum = 400 W, Dr. Hielscher GmbH, Germany). This ultrasonic transducer converts applied electrical waves into ultrasonic waves. In addition, the converter vibrated and transmitted at a constant frequency (24 kHz). This motion was applied to a cylindrical titanium sonotrode horn tip (model H14, 14 mm in diameter, Dr. Hielscher GmbH, Germany). The samples were treated by ultrasound. Apart from the special ultrasound conditions mentioned in the results, the general ultrasound conditions were as follows: The sonotrode horn tip was placed 20 mm from the bottom of surface of a beakers. Experimental setup for the preparation of nanoemulsions was performed. In addition, temperature measurements were controlled and it was stable by the use of an ice bath.
2.3 Ultrasonic intensity
The generated ultrasonic intensity from the sonotrode horn tip was calculated according to the formula stated below:
2.4 Nanoemulsion formation
In order to obtain oil in water emulsions (O/W) at ambient temperature, the designated amounts of clove oil, water and mixture of 5 wt% surfactants (Span® 80/Tween® 80) were blended. The samples were prepared by ultrasound method. A frequently used method for the selection of surfactants as emulsifying agents is known as hydrophilic–lipophilic balance (HLB) method. The HLB number of mixed surfactant system was calculated by the following equation:
In application of pesticide durability is very essential; therefore oil is added to stabilize the pesticide. Use of diluted pesticide (80–98% water) is customary and economically feasible in protection of agricultural products. Selection of 2.5–15% clove oil was on purpose for measurement of effective concentration on specific pest. The final ingredients for preparation of nanoemulsion of clove oil used for assessment of droplet stability in long duration (180 days) are summarized in Table 1.
| Concentration of clove oil (wt%) | Concentration of Tween® 80/Span® 80 (wt%) | Content of water (wt%) |
|---|---|---|
| 2.5 | 5.0 | 92.5 |
| 5.0 | 5.0 | 90.0 |
| 7.5 | 5.0 | 87.5 |
| 10.0 | 5.0 | 85.0 |
| 15.0 | 5.0 | 80.0 |
2.5 Measurement of droplet size
The mean droplet size and size distribution of clove oil nanoemulsions was determined by dynamic light scattering using a Zetasizer® Nano Series (Nano ZS model ZEN 3600, Malvern, UK) at a fixed scattered angle of 173°. Measurements were made at 25 °C and each measurement was performed for three times. The software used to collect and analyze the data was the Zetasizer® Software (version 7.03). All experiments were performed in triplicates and the average obtained values are illustrated in Figs. 1–4.
Effect of duty cycle on the droplets size of clove oil nanoemulsion using ultrasound method, means ± SD (n = 3).
Effect of ultrasonic intensity on the droplets size of clove oil nanoemulsion using ultrasound method, means ± SD (n = 3).
Effect of ultrasonication time on the droplets size of clove oil nanoemulsion using ultrasound method, means ± SD (n = 3).
Effect of clove oil content on the droplets size of clove oil nanoemulsion using ultrasound method for duration of six months, means ± SD (n = 3).
3 Results and discussion
3.1 Effect of duty cycle of ultrasonic exposure on droplets size of clove oil nanoemulsion
The effect of duty cycle in production of clove oil nanoemulsion at width of pulse of 0.25%, 0.5%, 0.75% and 1% was investigated. Fig. 1 shows the average of droplets size of clove oil nanoemulsion significantly decreased with an increase in duty cycle from 0.25% to 0.75%. As the duty cycle increased from 0.75% to 1%, there were no significant changes in droplets size. It was indicated that cavitation effect of pulse ultrasound as duty cycle 0.75% was better than duty cycle of 1%; because both had the same droplets size but, duty cycle of 0.75% had lower energy consumption. Also, when the duty cycle increased, the intensity of cavitation increased the force applied to the disintegration of large droplets. In duty cycle of 0.75%, the sample droplets size became small (94.7 ± 6.7 nm) and transparent. Therefore for the rest of experiments the ultrasonic duty cycle was fixed at 0.75%.
3.2 Effect of ultrasonic intensity
The stable clove oil emulsions with various input ultrasonic intensities were prepared with 10 wt% of clove oil, 5 wt% of Tween® 80/Span® 80 surfactants at HLB 9 (according to Eq. (1), mixed surfactants used 56 wt% of Span® 80 and 44 wt% of Tween® 80), cycle 0.75% and ultrasonication time 450 s. Fig. 2 shows the average of droplets size of clove oil nanoemulsion decreased with an increase in ultrasonic intensity ranging from 52 to 259 W/cm2. Increase in ultrasonic intensity, caused an increase in shear stress; that can be explained with a great number of cavitation tiny bubbles which was created in the liquid and the bubbles collapsed drastically, so, clove oil droplets size was small. However, decrease in the droplets size, depends on the ability and performance of surfactants. As long as the amount of emulsifier is sufficient to cover the newly formed droplets, with an increase in the amount of energy used in the course of homogenization process, the droplets size is decreased. The droplets size at ultrasonic intensities of 208 and 259 W/cm2 did not change considerably (around 50 nm). In addition, when the input ultrasonic intensity increases, it might increase drops movement and it seems drops coalescence speed more quickly drops covered by the surfactants. During the experiment the ultrasonic intensity was fixed at 208 W/cm2.
3.3 Effect of ultrasonication time
The effect of ultrasonication time on the droplets size of clove oil nanoemulsion was determined. Fig. 3 shows the stable clove oil nanoemulsions prepared by ultrasonication method with various ultrasonication times. Operating conditions were∗ 10 wt% of clove oil, 5 wt.% of Tween® 80/Span® 80 surfactants at HLB 9, cycle 0.75% and ultrasonic intensity was 208 W/cm2. Sonication time has considerable effect on droplet size. Increase in sonication time could be due to an increase in shear forces applied on the droplets which cause more deformation and more fragmentation of the droplets. It is expected to have small droplets size with increasing sonication time.
However, illustrated results in Fig. 3 show droplets size within 300 s became nearly the smallest droplets size and it wasn’t significantly reduced at high sonication time. The droplets size tiny decreased (around 10 nm) gradually as sonication time increased from 300 to 900 s and then it did not continue to increase within the time range. According to the investigation, the cavitation effect of ultrasound, including macro-turbulence created by the implosion of cavitation micro-bubbles and microjets generated by the cavitation on the clove oil after 300 s; that was just wasted energy. Therefore, it was decided that during the experiment the ultrasonic time should be fixed at 300 s.
3.4 Effect of clove oil content on nanoemulsions for duration of 6 months
The effect of clove oil content on the droplets size of nanoemulsion was investigated on the first day with increasing clove oil content from 2.5, 5, 7.5, 10 and 15 wt%. All emulsions made by ultrasound with optimum formula conditions were mixed with ultrasonication time of 300 s, surfactants concentration of 5 wt%, HLB number equal to 9, duty cycle 0.75% and ultrasonic intensity 208 W/cm2. Fig. 4 shows in most cases the emulsions were formed with the smallest diameter at the first day; for clove oil content of 2.5%, 5%, 7.5%, 10% and 15% as nanoemulsions droplets size was 145.3 ± 11.5, 63.3 ± 10.0, 47.3 ± 3.8, 43.3 ± 5.7 and 36.0 ± 6.2 nm, respectively. Then the physical stability of clove oil nanoemulsions with various levels of clove oil content was studied for 180 days at a storage room. After 7, 30, 90 and 180th day of making clove oil nanoemulsions with various clove oil content, they were re-sized, as shown in Fig. 4, all nanoemulsion made by optimum formula had well physical stability during storage time. In addition, the constant droplets size and slight increase in the droplets size in some cases are a sign of their physical stability during storage time for duration of 30 days. In 90th day, all emulsions, except the first one (with 2.5% clove oil content) had slightly increased in droplets size; while, the developed size of droplet was within the range of defined nanoemulsion droplets size. Finally, after 180 days, the prepared nanoemulsions showed the modest changes in droplets size. Furthermore, the minimum droplets size nanoemulsions were stabled with 7.5 and 10 wt.% of clove oil with average droplets size about 100 nm.
In fact, Ostwald ripening is the phenomenon that occurs in nanoemulsions in which the oil phase may be slightly soluble within the surrounding aqueous phase (Tadros et al., 2004). For long duration (180 days) the droplets size slightly increased, that was most probably due to interaction nanoemulsion molecules and also based on collision theory the small droplets are collided and developed a reasonable large droplets size. The mean droplets size measurement for the clove oil nanoemulsions for duration of 180 days proved that the droplet size gradually increased. Fortunately, there was no phase separation after long duration (180 days). The droplet size was quite stable and even increasing droplet size after long duration was certainly not affecting on properties of nanoemulsion.
4 Conclusions
Based on evaluation of effective parameters in preparation of nanoemulsion, present research provides very useful information for the production of ultrasonication emulsification of clove oil as a green pesticide. The results obtained in this research showed that the discussed factors of ultrasonication influenced in decrease of average droplets size of clove oil nanoemulsion under ultrasound emulsification. The droplets size of clove oil nanoemulsion was a function of duty cycle, ultrasonic intensity, ultrasonication time and clove oil ratio. Suitable ultrasonic intensity was necessary to produce clove oil nanoemulsion. In addition, pulsed ultrasound at specific intervals could be more efficient than continuous sonication with the advantage of saving energy consumption. Ultrasound process conditions were defined with various clove oil content (2.5–15 wt%), ultrasonication time (300 s), surfactants concentration of 5 wt%, HLB number 9, duty cycle 0.75% and ultrasonic intensity 208 W/cm2 for the duration of 6 months. These findings demonstrate that the use of ultrasonication emulsification method has considerable advantages for production of clove oil nanoemulsion as a green pesticide with the nanoemulsion droplet size of 43 nm. At ambient temperature, this formulation can be stable for duration of six months with average droplets size about 100 nm. Therefore, more attention should be paid in application of ultrasound technique for the production of nanopesticide.
Acknowledgments
The present research is a part of PhD thesis in Chemical Engineering, Biotechnology. This research was supported by Nanotechnology Research Institute, Babol Noshirvani University of Technology, Iran, and the Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark.
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