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Preparation and characterization of Mentha × piperita oil emulsion for housefly (Musca domestica L.) control
Industrial Crops and Products 44 (2013) 611–617
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Preparation and characterization of Mentha × piperita oil emulsion for housefly (Musca domestica L.) control Peeyush Kumar, Sapna Mishra, Anushree Malik ∗ , Santosh Satya Applied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology Delhi, New Delhi 110 016, India
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Article history: Received 8 June 2012 Received in revised form 11 September 2012 Accepted 15 September 2012 Keywords: Mentha × piperita Emulsion Characterization Bioefficacy Musca domestica
a b s t r a c t Emulsified concentrate (EC) formulation of Mentha × piperita oil was prepared by taking (w/w) 40% oil, 45% aeromax, 3% butanol-1 and 12% surfactant (CABS-70 and NP-20 in variable ratio). Three ECs (designated as A, B and C) were screened amongst prepared ECs on the basis of physical criteria. Emulsions were assessed for their stability through creaming volume, particle size and zeta potential determination. Particle size of emulsions varied between 536.7 and 1133.6 nm, while zeta potential value (−45.9 to −47.3 mV) was strongly negative suggesting stability. Characterization of emulsion for its pH (≈6.6–7.5) and flash point analysis (≈90–92 ◦ C) indicated a stable formulation, which was safe to handle and transport. Prepared emulsions were stable at various temperatures (4–60 ◦ C), and showed a thin creaming layer at the upper surface without any phase separation, when subjected to centrifugation. Freeze/thaw cycles showed no visible change in appearance in EC ‘A’ and ‘B’, while EC ‘C’ showed tendency to rupture, i.e. dispersion of oil droplets in emulsion, after 6 cycles. Bioefficacy of EC ‘B’, adjudged as most stable emulsion, against adults of housefly (Musca domestica) showed 70–88% repellency with freshly prepared EC. The repellency decreased to 34–72% after 12 months storage at room temperature. Assay against housefly larvae showed an LC50 value of 0.08 L/cm2 (emulsion from freshly prepared EC) and 0.34 L/cm2 (emulsion from stored EC). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Mentha × piperita oil is known for its medicinal, aromotherapeutic and anti-cancerous properties (Kumar et al., 2011a). Its bioactivity against bacteria, fungus and insects are well established (Bakkali et al., 2008). However, its practical application warrants a cost effective and stable formulation with ability to cater wide range of function. Emulsion, in this regard, with numerous applications, in foods, cosmetics, medicine, paints, metal and wood processing (Chen and Tao, 2005), presents a suitable candidature. For, insect control, emulsion formulation of essential oils has an advantage of wide sprayability, making it suitable for varied application against different stages of insects inhabiting diverse habitats (Kumar and Parmar, 2000). Besides this, emulsion often improves the biological activity of active ingredients (Moretti et al., 2002). Simple preparation technology and low production cost adds to its appeal as formulation of choice for developing countries that face the insect menace due to poor sanitation and hygiene.
∗ Corresponding author. Tel.: +91 11 26591158; fax: +91 11 26591121. E-mail addresses: [email protected], anushree [email protected] (A. Malik). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.09.013
The emulsion formulation of essential oils has been attempted earlier in some studies (Edris, 1998; Kumar and Parmar, 2000; Varona et al., 2009; Bylaite et al., 2001; ElShafei et al., 2010; Carrillo-Navas et al., 2012; Rodríguez-Rojo et al., 2012). Edris (1998) evaluated effect of stabilizer on orange oil emulsion, while same was assessed for caraway essential oil by Bylaite et al. (2001). Similarly, effect of different surfactants on stability of rosemary essential oil has been evaluated by Rodríguez-Rojo et al. (2012). Carrillo-Navas et al. (2012) investigated the rheological properties of chia essential oil. Effect of process variables (operation time, homogenization velocity and surfactant concentration) on the physical properties and stability of lavandin oil emulsion (drop diameter and creaming velocity) has been studied by Varona et al. (2009). Kumar and Parmar (2000) prepared 33 recipes of neem oil based emulsified concentrate (EC) and evaluated its bioefficacy against larvae of the Bihar hairy caterpillar, Spilosoma obliqua Walker. Similarly, Moretti et al. (2002) investigated the effects of emulsion of different essential oils against larvae of gypsy moth, Limantria dispar, and reported variation in emulsion efficacy with variation in essential oils. From the above discussion, it is apparent that, most of the studies have limited their objectives to emulsion characterization, while activity or efficacy of prepared formulations was seldom evaluated. Further, studies attempting vector control with essential oil emulsions were even less (Kumar and
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Parmar, 2000; Moretti et al., 2002). Shelf life analysis of emulsion, which forms a very crucial parameter to determine its usability and efficacy, also remains neglected. Moreover, no study investigating Mentha oil emulsion could be retrieved. In view of the above, the present study has developed M. × piperita oil emulsion for housefly control. Earlier, the essential oil of M. × piperita has been found to be most effective for housefly control in the various assays done in our lab (Kumar et al., 2011b, 2012). The present study evaluates the effect of homogenization and variable surfactant concentration on emulsion. Further bioassay against housefly as well as the shelf life of selected emulsion was also conducted. 2. Material and methods In the present study initially several variants of emulsified concentrate (EC) formulation of M. × piperita oil were prepared. Initially, the surfactants for emulsion formation were chosen on the basis of their HLB value. Further, optimization of surfactants concentration was based on trial and elimination, where variable ratio of two surfactants was tested, to chose best three of them. The chosen emulsion samples were assessed for the effect of homogenization on their properties. Prepared emulsion formulation was characterized for its creaming volume, particle size, zeta potential, pH, viscosity and flash point analysis, as well as subjected to the effect of various process parameter as; temperature, centrifugation and free-thaw properties. Bioefficacy of M. × piperita oil emulsion (freshly prepared and 12 month stored) was assessed against adults and larvae of Musca domestica to ascertain its role in insect control.
temperature (25 ± 2 ◦ C) and visually examined. The height of visible creamy layer (HC ) and height of emulsion (HE ) was recorded. Relative creaming layer was determined as: Relative creaming layer =
HC × 100 HE
2.3.2. Determination of droplet size Droplet size distribution of the M. × piperita oil emulsion was determined by Dynamic Light Scattering (Mastersizer 2000, Malvern instruments). Sampling was carried out with 0.01% emulsion, freshly prepared in double distilled water. High dilution of emulsion was used to prevent multiple scattering effects. Particle size measurements are reported as normalized intensity distribution as defined as the average emulsion diameter. Each sample was analyzed twice with each analysis consisting of three replicates. 2.3.3. Zeta potential The zeta potential was measured by a Zetasizer (Malvern Instruments). The measurements were carried out with 0.01% emulsions, in fully automatic mode. Each sample was analyzed twice, each analysis consisting of three replicates. 2.4. Characterization of EC formulation EC formulations prepared after third homogenization cycle were evaluated for different parameters to characterize their properties. Studies of different parameters of the emulsions give hints to the stabilization mechanism of the emulsifier/co-emulsifier system.
2.1. Materials The essential oil of M. × piperita was purchased from Kanta Chemicals Pvt. Ltd., Khari Bowli, New Delhi, India and stored in plastic bottles at 4 ◦ C. Surfactants; calcium alkyl benzene sulponate (CABS-70) and nonyl phenol (NP-20) were obtained from Standard Surfactant Ltd. (Kanpur, India) and Magnan, respectively. Aeromax and Butanol-1 were purchased from Global Tech and Hi-Media India Ltd., respectively. 2.2. Emulsion preparation Emulsified concentrate (EC) formulation of M. × piperita oil was prepared by taking (w/w) 40% oil, 45% aeromax, 3% butanol-1 and 12% surfactant. Two surfactants, CABS-70 and NP-20 were used in the variable ratio (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to prepare ECs of three different surfactant concentrations. Prepared ECs were diluted to form 2% emulsion and scanned for creaming volume (the appearance of an oil phase at the surface of the system), sedimentation and color. On the basis of these physical criterions, three ECs, designated as A, B and C (CABS-70:NP-20; 40:60, 50:50, 30:70, respectively) were subjected to three cycle of homogenization (each cycle of 5000 rpm for 30 min) as further preparative steps. 2.3. Effect of homogenization Effect of each homogenization cycle on all the three ECs was evaluated in terms of its stability by determining creaming volume, particle size and zeta potential. 2.3.1. Determination of creaming volume of emulsions Creaming kinetics of each ECs were evaluated by preparing 2% emulsion in double distilled water. 100 mL of each emulsion were transferred to a glass cylinder with stopper and kept for 2 h at room
2.4.1. pH and conductivity measurement An HI 2212 pH meter (Hannah instruments, USA) was used for the determination of the pH value of the 2% emulsions at room temperature (25 ± 2 ◦ C). Conductivity of emulsion (2%) was measured by HI 9835 conductivity meter (Hannah instruments, USA) at room temperature (25 ± 2 ◦ C). 2.4.2. Viscosity analysis Viscosity was measured with a Brookfield Viscosimeter (Brookfield, USA). About 40 mL of EC was heated at 25 ± 2◦ C in the measuring cylinder. The same spindle (spindle S61) and the same stirring rate (30 rpm) were used for each experiment. 2.4.3. Flash point analysis Flash point and fire point of ECs were calculated to define their flammability hazards. The flash and fire point was determined by the tag open-cup method by continuing the heating of the ECs to its fire point (ASTM D1310). 2.5. Effect of process parameters 2.5.1. Effect of temperature Each ECs were filled in cylindrical tubes (height 9 cm, dia. 2.5 cm) and were kept at 6 different temperature variations; 4 ◦ C, 15 ± 2 ◦ C, 30 ± 2 ◦ C, 45 ± 2 ◦ C and 60 ± 2 ◦ C. Every week samples were evaluated for color and creaming volume of emulsion (2%), till 6th week. 2.5.2. Effect of centrifugation Effect of centrifugation was measured with 10 mL of 2% emulsions at a centrifugal acceleration of 10,000 rpm for 20 min and 4 ◦ C (Sigma centrifuge, Germany). Each sample was subjected to 3 centrifugation cycles and after each cycle the creaming volume was measured.
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2.5.3. Freeze/thaw cycles Each EC was filled in cylindrical tubes (height 9 cm, dia. 2.5 cm) and stored for 24 h in a freezer at −21 ◦ C and then for 12 h at room temperature (25 ± 2 ◦ C). This cycle was repeated six times. After each cycle, 2% emulsion was prepared and any change in creaming volume and sedimentation was observed. 2.6. Bioassay against housefly Based on the above characterization, best emulsion was chosen for its bioassay against housefly adult (repellency assay) and larvae (larvicidal assay). The assay was done both with freshly prepared emulsion and with emulsion stored for 12 months at ambient temperature (varying between 10 ± 5 ◦ C and 40 ± 5 ◦ C). 2.6.1. Repellency assay The repellent effects of M. × piperita oil formulation was evaluated against field-collected adult houseflies (n = 50) in a repellency chamber (Kumar et al., 2011b). Repellency chamber consisted of an outer chamber (20 cm × 20 cm × 20 cm) which was connected to an inner chamber (60 cm × 60 cm × 60 cm) by a hole measuring 9 cm × 9 cm. Outer chamber contained a Petri plate (dia. – 9 cm) containing filter paper uniformly impregnated with test solutions of emulsion formulation. Three different dilutions of M. × piperita oil emulsion, 1, 3, and 5%, corresponding to the concentration of 0.003, 0.009 and 0.016 L/cm2 was used as test solution. In the control treatment, the paper was treated with a mixture of aromax and surfactant. The experiment was performed for 4 h, at the end of which the number of flies repelled from outer to inner chamber was counted to determine the percentage repellency. Three replicates of each treatment were performed. 2.6.2. Larvicidal bioassay Larvicidal bioassay was performed with lab reared housefly larvae. Larvae (20, 2nd instar) were placed on a filter paper containing a diet of 2 g groundnut oil cake, 5 g wheat bran, 2 g milk powder and 1 g honey mixed with 10 mL water. In a pour-on treatment, 500 L of M. × piperita oil emulsion at the dilutions of 1%, 3% and 5% in water, corresponding to oil concentration of 0.003, 0.009 and 0.016 L/cm2 was added to the diet. Diet material in the control treatment was sprayed with a mixture of aromax and surfactant. Three replicates of each oil treatment were performed. Larvae were observed for any change in appearance and mobility over the subsequent 48 h. Larval mortality was assessed by withering and the development of a brownish appearance (Kumar et al., 2011b). 2.7. Statistical analysis Mortality data were corrected for control mortality according to Abbott (1925) and normalized using arcsine transformation. Data for 50% and 90% lethal concentration for larval mortality (LC50 and LC90 ) were calculated using SPSS (2007) software. 3. Results and discussion 3.1. Effect of homogenization Effect of homogenization on emulsion was studied in relation to its outcome on relative creaming volume, particle size and zeta potential. Effect of homogenization on relative creaming volume showed decrease in creaming layer thickness with each homogenization cycle (Fig. 1). For each EC, creaming volume decreased by more than half with each homogenization cycle; with no creaming layer being observed in EC ‘B’, after 2nd homogenization
Fig. 1. Effect of homogenization on relative creaming volume of emulsified concentrate (EC).
cycle. Creaming volume has been used as stability criteria for orange oil emulsion and is reported to be influenced by surfactant concentration (Edris, 1998). Effect of homogenization on particle size of ECs is represented in Fig. 2. A slight decrease in average particle size with each homogenization cycles was observed for EC ‘B’ (Fig. 2B). Similarly, the particle size of EC ‘C’ after 1st, 2nd and 3rd homogenization cycles was 4207.8 nm, 3684 nm, and 1133.6 nm, respectively. Homogenization assists emulsion formation by providing mechanical energy, which helps in emulsion stability by counteracting particle coalescence (Chen and Tao, 2005). Hence, homogenization is expected to decrease the average particle size. However, the average particle size of EC ‘A’ showed variability with homogenization cycles; being 345.9 nm (after 1st homogenization cycle), 852.6 nm (after 2nd homogenization cycle) and 563.7 nm (after 3rd homogenization cycle) (Fig. 2A). The increase of droplet size reflected an ongoing coalescence process inside the emulsion, thus decrease in stability. The apparent disparity in particle size of emulsion with homogenization procedure is probably due to effect of molecular properties of surfactant, surfactant–solvent interaction and self-aggregated structure formed by surfactants molecules, which has significant role in determining particle size and polydispersity of emulsion droplets (Rodríguez-Abreu and Lazzari, 2008). In the present study, EC ‘B’ had equal concentration of both the surfactants, while the concentration of surfactants (CABS-70:NP-20) was more skewed for EC ‘A’ (0.7) and EC ‘C’ (0.5). The variation in surfactant concentration is reflected in particle size of emulsions, in the present study. Also, homogenization effect on emulsion stability is apparent, particularly for EC ‘C’. Roland et al. (2003) also reported variation in median size of the emulsion with surfactant concentration and homogenization cycle, while Varona et al. (2009) observed homogenization velocity to have greatest impact on emulsions’ particle size. Zeta potential measurement for different ECs with homogenization cycles are presented in Fig. 3. All the values of zeta potential were strongly negative and lied between −43 mV and −54 mV, reflecting emulsion stability. Zeta potential measures the repulsive force created by surfactant adsorption on the surface of dispersed phase droplet of emulsion, thus characterizes deflocculation capacity of emulsion and reflects its stability (Roland et al., 2003). Although, a general decrease in value of zeta potential was observed with each homogenization cycle, the changes were not as significant as in the case of creaming volume and particle size. The above results showed the significance of homogenization cycle on stability of emulsion with varied surfactant concentrations. Also, the optimization of surfactant concentration is crucial to produce a stable end product as reflected by values of creaming volume, particle size and zeta potential (Dickinson, 1992).
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Fig. 2. Particle size of emulsified concentrate (EC): (A) EC ‘A’, and (B) EC ‘B’, after (a) 1st, (b) 2nd, and (c) 3rd homogenization cycles.
Fig. 3. Zeta potential of emulsified concentrate (EC): (A) EC ‘A’, (B) EC ‘B’, and (C) EC ‘C’, after (a) 1st, (b) 2nd, and (c) 3rd homogenization cycles.
3.2. Characterization of EC formulations The properties of different emulsions prepared with varied concentrations of two surfactants; CABS-70 (anionic) and NP-20
(non-anionic) are shown in Table 1. All the ECs were reddish in appearance. ECs A and B exhibited slightly acidic pH while EC ‘C’ had slightly alkaline pH. Viscosity of EC ‘A’, ‘B’ and ‘C’ were 25, 28.5 and 30 cP, respectively. Viscosity of emulsion is proportional
Fig. 4. Effect of temperature on emulsified concentrate (EC ‘B’).
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Fig. 5. Effect of storage temperature on relative creaming volume of emulsified concentrates (ECs).
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Fig. 6. Effect of centrifugation on relative creaming volume of emulsified concentrates (ECs).
to volume of dispersed phase, and is also influenced by surfactant concentration (Tadros, 1994). The flash point is a measure of the tendency of a sample to form flammable mixtures with air in controlled laboratory conditions and is parameter for storage and handling consideration of flammable materials (Encinar et al., 2005). Flash point of ECs varied between 90 and 92 ◦ C, which was approximately equivalent to flash point of M. × piperita essential oil (92 ◦ C). The flash point of formulation obtained in this study, was much higher than the prescribed minimum limit of 24.5 ◦ C (Kumar and Parmar, 2000). Relatively high flash point of the product observed in this study, makes it less volatile and safer to transport and handle. Particle size of EC ‘B’ was observed to be lowest at 223.5 nm. After a storage period of 12 months, average particle size of emulsion showed an increment of 31–48%, probably due to agglomeration of emulsion droplets. Creaming volume measurement of stored emulsion also showed significant increase (Table 1).
stability of EC ‘B’ which showed relative creaming volume of 2%, after 3rd centrifugation cycle. Minimum stability was found in EC ‘C’, having relative creaming volume of 6%, after 3rd centrifugation cycle. The stability of ECs under centrifugation pressure reflects the strength of the interfacial film, thus stability of emulsion (Puisieux and Seiller, 1983). Freeze/thaw cycles showed no visible change in appearance in EC ‘A’ and ‘B’, while EC ‘C’ showed tendency to rupture, i.e. dispersion of oil droplets in emulsion, after 6 cycles. The result obtained in this study was better than Roland et al. (2003) who reported rupturing of the emulsions after five cycles. Similarly, in the study by Kumar and Parmar (2000) cold treatment of neem oil ECs at 0 ◦ C showed turbidity and slight solid separation. Overall, the various physical properties of ECs showed a correlation, with one property complimenting another. This could be comprehended for a particular EC (or emulsion), where value of all the three indicated properties (creaming volume, particle size and zeta potential) pointed to their stability. For EC ‘B’, the value of their creaming volume and particle size, both signified their better stability compared to EC ‘A’ or EC ‘C’. Results of various process variables also indicated EC ‘B’ as most stable to any external changes. The difference in stability for ECs could be attributed to the variation in surfactant concentration. As is evident from the results, EC containing equal ratio of surfactants, CABS-70:NP-20 (EC ‘B’) was most stable, while stability showed propensity to decrease with the skewing of surfactant ratio away from its mid point.
3.3. Effect of process parameters Storage of ECs at different temperature demonstrated yellowish-reddish-dark reddish appearance (Fig. 4). Relative creaming volumes of emulsions at various temperatures are shown in Fig. 5. Highest relative creaming volume was seen in EC ‘C’ at different temperatures, while EC ‘A’ and EC ‘B’ showed comparatively thin creaming layer. Emulsions were stable at the temperature of 4, 15, 30 ◦ C and to some extent even at 45 ◦ C. At the temperature of 60 ◦ C, phase separation in emulsion was observed, and thus was not considered suitable for emulsion stability during storage. The result obtained in this study was compatible with the one obtained by Edris (1998) who reported increase in creaming layer of orange oil emulsion with increase in temperature, and obtained lowest creaming volume at 4 ◦ C. Centrifugation is used to assess the stability of emulsion by speeding up potential destabilization process such as, creaming and sedimentation. Effect of centrifugation on emulsions is shown in Fig. 6. Emulsion showed thin creaming layer at the upper surface without any phase separation. The figure represents maximum
3.4. Bioassay against housefly On the basis of various characterizations EC ‘B’ was adjudged as best, being most stable and having lower sedimentation tendency. Thus, EC ‘B’ was chosen for bioassay study against housefly adults and larvae. Rates of repellency of adult houseflies by different concentration of M. × piperita oil emulsion are shown in Fig. 7. Emulsion prepared from freshly prepared EC showed 70–88% repellency at the end of 4 h of observation, which decreased to 34–72% of repellency for emulsion prepared from EC formulation stored for 12 months at
Table 1 Properties of three emulsified concentrate (EC) formulation of M. × piperita oil. Properties
A
B
C
pH Electrical conductivity (S/cm) Flash point (◦ C) Fire point (◦ C) Viscosity (cP) Particle size (nm) Zeta potential (mV) Particle size after 12 month storage (nm) Relative creaming volume (%) Relative creaming volume after 12 month storage (%)
6.6 88 92 102 25 536.7 −45.9 712.2 0.4 3.7
6.65 47.7 91 101 28.3 223.5 −43.8 293.9 0 2.1
7.54 30.8 90 101 30 1133.6 −47.3 1680.0 0.8 7.7
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Table 2 Lethal concentration (LC50 and LC90 ) and lethal time (LT50 ) of M. × piperita oil emulsions against 2nd instar larvae of Musca domestica. Mentha oil emulsion
Dilution (%)
Concentration (L/cm2 )
LT50 (h)
LC50
After 0 month
1 3 5
0.003 0.009 0.016
17.1 11.3 9.8
0.08
0.20
After 12 month
1 3 5
0.003 0.009 0.016
59.3 52.7 40.4
0.34
0.49
[12 h]
(L/cm2 )
LC90
[12 h]
(L/cm2 )
In their study, the crude form of essential oil showing higher efficacy (Thymus herba-barona essential oil), maintained its activity in formulation. The discussion from the above literature studies also opines the significance of oil and surfactant selection and preparation methodology on emulsion properties and also, on emulsion bioefficacy. In context of housefly control, emulsion of essential oil being superior spray carrier, could play significant role for the control of gregarious adult flies. Also, emulsion formulations are excellent for application against insects which tend to hide through opening or cervices, viz. stored grain pests, housefly larvae. However, in view of the absolute paucity of literature pertaining to essential oil emulsions for housefly control, the current report makes a pioneering contribution. Fig. 7. Repellency (%) of Musca domestica adults by M. × piperita oil emulsion at ) indicate emulsion from freshly prepared different concentration [solid lines ( emulsified concentrate; broken lines ( ) indicate emulsion from emulsified concentrate stored for a period of 12 months; 0.016 L/cm2 ; 0.009 L/cm2 ; 䊉 0.003 L/cm2 ].
the room temperature. Larvicidal bioassay for emulsion from both freshly prepared and stored EC showed significant variation with doses applied, time of observation and interaction between doses and time. Lethal concentration, LC50 for emulsion from freshly prepared EC was 0.08 L/cm2 while emulsion of stored EC showed LC50 value of 0.34 L/cm2 (Table 2). The results obtained from the present study indicates slight loss in efficacy of EC formulation over storage period, insecticidal properties of formulation was not lost completely, even after storage period of 12 months. Hence, enhanced dosage of the stored EC formulation would be required for comparable efficacy. The formulation was stored at room temperature, with ambient storage temperature fluctuating between 10 ± 5 ◦ C and 40 ± 5 ◦ C. Significant temperature variations could have lead to the loss in bioactivity. The depreciation in emulsion bioefficacy could also be attributed to reduction in its stability due to agglomeration of oil droplets over storage period as reflected by the particle size and creaming volume analysis (Table 1). However, more than 90% of the oil remained in the emulsified form for all the emulsions. Compared to the result of present study, lavandin essential oil has been reported to retain 80% of the oil in the stabilized form after 50 days of storage at 5 ◦ C (Varona et al., 2009). Therefore, the stability results obtained in the present study for Mentha oil emulsion, pertaining to storage for 1 year at ambient temperature, is quite encouraging. Further improvement in shelf life can be achieved by storage under controlled temperature conditions and the investigations on these lines are underway. As discussed earlier, only few studies could be retrieved on essential oil formulations for insect control. Kumar and Parmar (2000) prepared and evaluated neem oil based emulsions against 2nd instar larvae of hairy caterpillar, Spilosoma obliqua. In their study, they deduced the dependence of emulsion activity on surfactant types and concentration. In another study, Moretti et al. (2002) reported variation in emulsion bioefficacy against larvae of gypsy moth, Limantria dispar, with variation in essential oils.
4. Conclusions In an attempt to utilize biological control potential of M. × piperita oil, an emulsion formulation was prepared. Preparation methodology highlighted the variation in emulsion properties (creaming volume, particle size and zeta potential) with homogenization cycle and surfactant concentration. Characterization of emulsion for its pH, viscosity and flash point analysis indicated a stable formulation, which was safe to handle and transport. Further, stability of emulsion was established at various process parameters (temperature, centrifugation and free-thaw properties). Bioefficacy of emulsion against adults and larvae of housefly was found to be appreciable at various dilution, but showed slight decrease in efficacy with storage period. Acknowledgments The authors acknowledge Mr. Jai Kumar (Formulation Division, IPFT, Gurgaon) and Mr. Sabal Singh (IIT Delhi, India) for their help in experimental work. Partial financial support from KVIC Interface project is gratefully acknowledged. References Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46, 446–475. Bylaite, E., Nylander, T., Venskutonis, R., Jönsson, B., 2001. Emulsification of caraway essential oil in water by lecithin and b-lactoglobulin: emulsion stability and properties of the formed oil–aqueous interface. Colloids Surf. B: Biointerfaces 20, 327–340. Carrillo-Navas, H., Cruz-Olivares, J., Varela-Guerrero, V., Alamilla-Beltrán, L., VernonCarter, E.J., Pérez-Alonso, C., 2012. Rheological properties of a double emulsion nutraceutical system incorporating chia essential oil and ascorbic acid stabilized by carbohydrate polymer–protein blends. Carbohydr. Polym. 87, 1231–1235. Chen, G., Tao, D., 2005. An experimental study of stability of oil–water emulsion. Fuel Process. Technol. 86, 499–508. Dickinson, E., 1992. Interfacial interactions and the stability of oil-in water emulsions. Pure Appl. Chem. 64, 1721–1724. Edris, A.E., 1998. Preparation and stability of a protein stabilized orange oil-in-water emulsion. Nahrung 42, 19–22. ElShafei, G.M.S., El-Said, M.M., Attia, H.A.E., Mohammed, T.G.M., 2010. Environmentally friendly pesticides: essential oil-based w/o/w multiple emulsions for anti fungal formulations. Ind. Crops Prod. 31, 99–106.
P. Kumar et al. / Industrial Crops and Products 44 (2013) 611–617 Encinar, J.M., Juan, F., Gonzalez, J.F., Rodriguez-Reinares, A., 2005. Biodiesel from used frying oil: variables affecting the yields and characteristics of the biodiesel. Ind. Eng. Chem. Res. 44, 5491–5499. Kumar, L., Parmar, B.S., 2000. Effect of emulsion size and shelf life of azadirachtin A on the bioefficacy of neem (Azadirachta indica A. Juss) emulsifiable concentrates. J. Agric. Food Chem. 48, 3666–3672. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011a. Insecticidal properties of Mentha species: a review. Ind. Crops Prod. 34, 802–817. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011b. Repellent, larvicidal and pupicidal properties of essential oils and their formulations against the housefly, Musca domestica. Med. Vet. Entomol. 25, 302–310. Kumar, P., Mishra, S., Malik, A., Satya, S., 2012. Efficacy of Mentha × piperita and Mentha citrata essential oils against housefly, Musca domestica L. Ind. Crops Prod. 39, 106–112. Moretti, M.D.L., Sanna-Passino, G., Demontis, S., Bazzoni, E., 2002. Essential oil formulations useful as a new tool for insect pest control. AAPS Pharm. Sci. Tech. 3, 1–11.
617
Puisieux, F., Seiller, M., 1983. Galenica-Agents de surface et émulsions. Lavoisier, Paris. Rodríguez-Abreu, C., Lazzari, M., 2008. Emulsions with structured continuous phases. Curr. Opin. Colloid Interface Sci. 13, 198–205. Rodríguez-Rojo, S., Varona, S., Núnez, M., Cocero, M.J., 2012. Characterization of rosemary essential oil for biodegradable emulsions. Ind. Crops Prod. 37, 137– 140. Roland, I., Piel, G., Delattre, L., Evrard, B., 2003. Systematic characterization of oil-in-water emulsions for formulation design. Int. J. Pharm. 263, 85– 94. SPSS, 2007. Statistical product and service solution, system user’s guide, version 17.5. Tadros, T.F., 1994. Fundamental principles of emulsion rheology and their applications. Colloids Surf. A 91, 39–55. Varona, S., Martín, Á., Cocero, M.J., 2009. Formulation of a natural biocide based on lavandin essential oil by emulsification using modified starches. Chem. Eng. Proc. 48, 1121–1128.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Preparation and characterization of Mentha × piperita oil emulsion for housefly (Musca domestica L.) control Peeyush Kumar, Sapna Mishra, Anushree Malik ∗ , Santosh Satya Applied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology Delhi, New Delhi 110 016, India
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Article history: Received 8 June 2012 Received in revised form 11 September 2012 Accepted 15 September 2012 Keywords: Mentha × piperita Emulsion Characterization Bioefficacy Musca domestica
a b s t r a c t Emulsified concentrate (EC) formulation of Mentha × piperita oil was prepared by taking (w/w) 40% oil, 45% aeromax, 3% butanol-1 and 12% surfactant (CABS-70 and NP-20 in variable ratio). Three ECs (designated as A, B and C) were screened amongst prepared ECs on the basis of physical criteria. Emulsions were assessed for their stability through creaming volume, particle size and zeta potential determination. Particle size of emulsions varied between 536.7 and 1133.6 nm, while zeta potential value (−45.9 to −47.3 mV) was strongly negative suggesting stability. Characterization of emulsion for its pH (≈6.6–7.5) and flash point analysis (≈90–92 ◦ C) indicated a stable formulation, which was safe to handle and transport. Prepared emulsions were stable at various temperatures (4–60 ◦ C), and showed a thin creaming layer at the upper surface without any phase separation, when subjected to centrifugation. Freeze/thaw cycles showed no visible change in appearance in EC ‘A’ and ‘B’, while EC ‘C’ showed tendency to rupture, i.e. dispersion of oil droplets in emulsion, after 6 cycles. Bioefficacy of EC ‘B’, adjudged as most stable emulsion, against adults of housefly (Musca domestica) showed 70–88% repellency with freshly prepared EC. The repellency decreased to 34–72% after 12 months storage at room temperature. Assay against housefly larvae showed an LC50 value of 0.08 L/cm2 (emulsion from freshly prepared EC) and 0.34 L/cm2 (emulsion from stored EC). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Mentha × piperita oil is known for its medicinal, aromotherapeutic and anti-cancerous properties (Kumar et al., 2011a). Its bioactivity against bacteria, fungus and insects are well established (Bakkali et al., 2008). However, its practical application warrants a cost effective and stable formulation with ability to cater wide range of function. Emulsion, in this regard, with numerous applications, in foods, cosmetics, medicine, paints, metal and wood processing (Chen and Tao, 2005), presents a suitable candidature. For, insect control, emulsion formulation of essential oils has an advantage of wide sprayability, making it suitable for varied application against different stages of insects inhabiting diverse habitats (Kumar and Parmar, 2000). Besides this, emulsion often improves the biological activity of active ingredients (Moretti et al., 2002). Simple preparation technology and low production cost adds to its appeal as formulation of choice for developing countries that face the insect menace due to poor sanitation and hygiene.
∗ Corresponding author. Tel.: +91 11 26591158; fax: +91 11 26591121. E-mail addresses: [email protected], anushree [email protected] (A. Malik). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.09.013
The emulsion formulation of essential oils has been attempted earlier in some studies (Edris, 1998; Kumar and Parmar, 2000; Varona et al., 2009; Bylaite et al., 2001; ElShafei et al., 2010; Carrillo-Navas et al., 2012; Rodríguez-Rojo et al., 2012). Edris (1998) evaluated effect of stabilizer on orange oil emulsion, while same was assessed for caraway essential oil by Bylaite et al. (2001). Similarly, effect of different surfactants on stability of rosemary essential oil has been evaluated by Rodríguez-Rojo et al. (2012). Carrillo-Navas et al. (2012) investigated the rheological properties of chia essential oil. Effect of process variables (operation time, homogenization velocity and surfactant concentration) on the physical properties and stability of lavandin oil emulsion (drop diameter and creaming velocity) has been studied by Varona et al. (2009). Kumar and Parmar (2000) prepared 33 recipes of neem oil based emulsified concentrate (EC) and evaluated its bioefficacy against larvae of the Bihar hairy caterpillar, Spilosoma obliqua Walker. Similarly, Moretti et al. (2002) investigated the effects of emulsion of different essential oils against larvae of gypsy moth, Limantria dispar, and reported variation in emulsion efficacy with variation in essential oils. From the above discussion, it is apparent that, most of the studies have limited their objectives to emulsion characterization, while activity or efficacy of prepared formulations was seldom evaluated. Further, studies attempting vector control with essential oil emulsions were even less (Kumar and
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Parmar, 2000; Moretti et al., 2002). Shelf life analysis of emulsion, which forms a very crucial parameter to determine its usability and efficacy, also remains neglected. Moreover, no study investigating Mentha oil emulsion could be retrieved. In view of the above, the present study has developed M. × piperita oil emulsion for housefly control. Earlier, the essential oil of M. × piperita has been found to be most effective for housefly control in the various assays done in our lab (Kumar et al., 2011b, 2012). The present study evaluates the effect of homogenization and variable surfactant concentration on emulsion. Further bioassay against housefly as well as the shelf life of selected emulsion was also conducted. 2. Material and methods In the present study initially several variants of emulsified concentrate (EC) formulation of M. × piperita oil were prepared. Initially, the surfactants for emulsion formation were chosen on the basis of their HLB value. Further, optimization of surfactants concentration was based on trial and elimination, where variable ratio of two surfactants was tested, to chose best three of them. The chosen emulsion samples were assessed for the effect of homogenization on their properties. Prepared emulsion formulation was characterized for its creaming volume, particle size, zeta potential, pH, viscosity and flash point analysis, as well as subjected to the effect of various process parameter as; temperature, centrifugation and free-thaw properties. Bioefficacy of M. × piperita oil emulsion (freshly prepared and 12 month stored) was assessed against adults and larvae of Musca domestica to ascertain its role in insect control.
temperature (25 ± 2 ◦ C) and visually examined. The height of visible creamy layer (HC ) and height of emulsion (HE ) was recorded. Relative creaming layer was determined as: Relative creaming layer =
HC × 100 HE
2.3.2. Determination of droplet size Droplet size distribution of the M. × piperita oil emulsion was determined by Dynamic Light Scattering (Mastersizer 2000, Malvern instruments). Sampling was carried out with 0.01% emulsion, freshly prepared in double distilled water. High dilution of emulsion was used to prevent multiple scattering effects. Particle size measurements are reported as normalized intensity distribution as defined as the average emulsion diameter. Each sample was analyzed twice with each analysis consisting of three replicates. 2.3.3. Zeta potential The zeta potential was measured by a Zetasizer (Malvern Instruments). The measurements were carried out with 0.01% emulsions, in fully automatic mode. Each sample was analyzed twice, each analysis consisting of three replicates. 2.4. Characterization of EC formulation EC formulations prepared after third homogenization cycle were evaluated for different parameters to characterize their properties. Studies of different parameters of the emulsions give hints to the stabilization mechanism of the emulsifier/co-emulsifier system.
2.1. Materials The essential oil of M. × piperita was purchased from Kanta Chemicals Pvt. Ltd., Khari Bowli, New Delhi, India and stored in plastic bottles at 4 ◦ C. Surfactants; calcium alkyl benzene sulponate (CABS-70) and nonyl phenol (NP-20) were obtained from Standard Surfactant Ltd. (Kanpur, India) and Magnan, respectively. Aeromax and Butanol-1 were purchased from Global Tech and Hi-Media India Ltd., respectively. 2.2. Emulsion preparation Emulsified concentrate (EC) formulation of M. × piperita oil was prepared by taking (w/w) 40% oil, 45% aeromax, 3% butanol-1 and 12% surfactant. Two surfactants, CABS-70 and NP-20 were used in the variable ratio (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to prepare ECs of three different surfactant concentrations. Prepared ECs were diluted to form 2% emulsion and scanned for creaming volume (the appearance of an oil phase at the surface of the system), sedimentation and color. On the basis of these physical criterions, three ECs, designated as A, B and C (CABS-70:NP-20; 40:60, 50:50, 30:70, respectively) were subjected to three cycle of homogenization (each cycle of 5000 rpm for 30 min) as further preparative steps. 2.3. Effect of homogenization Effect of each homogenization cycle on all the three ECs was evaluated in terms of its stability by determining creaming volume, particle size and zeta potential. 2.3.1. Determination of creaming volume of emulsions Creaming kinetics of each ECs were evaluated by preparing 2% emulsion in double distilled water. 100 mL of each emulsion were transferred to a glass cylinder with stopper and kept for 2 h at room
2.4.1. pH and conductivity measurement An HI 2212 pH meter (Hannah instruments, USA) was used for the determination of the pH value of the 2% emulsions at room temperature (25 ± 2 ◦ C). Conductivity of emulsion (2%) was measured by HI 9835 conductivity meter (Hannah instruments, USA) at room temperature (25 ± 2 ◦ C). 2.4.2. Viscosity analysis Viscosity was measured with a Brookfield Viscosimeter (Brookfield, USA). About 40 mL of EC was heated at 25 ± 2◦ C in the measuring cylinder. The same spindle (spindle S61) and the same stirring rate (30 rpm) were used for each experiment. 2.4.3. Flash point analysis Flash point and fire point of ECs were calculated to define their flammability hazards. The flash and fire point was determined by the tag open-cup method by continuing the heating of the ECs to its fire point (ASTM D1310). 2.5. Effect of process parameters 2.5.1. Effect of temperature Each ECs were filled in cylindrical tubes (height 9 cm, dia. 2.5 cm) and were kept at 6 different temperature variations; 4 ◦ C, 15 ± 2 ◦ C, 30 ± 2 ◦ C, 45 ± 2 ◦ C and 60 ± 2 ◦ C. Every week samples were evaluated for color and creaming volume of emulsion (2%), till 6th week. 2.5.2. Effect of centrifugation Effect of centrifugation was measured with 10 mL of 2% emulsions at a centrifugal acceleration of 10,000 rpm for 20 min and 4 ◦ C (Sigma centrifuge, Germany). Each sample was subjected to 3 centrifugation cycles and after each cycle the creaming volume was measured.
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2.5.3. Freeze/thaw cycles Each EC was filled in cylindrical tubes (height 9 cm, dia. 2.5 cm) and stored for 24 h in a freezer at −21 ◦ C and then for 12 h at room temperature (25 ± 2 ◦ C). This cycle was repeated six times. After each cycle, 2% emulsion was prepared and any change in creaming volume and sedimentation was observed. 2.6. Bioassay against housefly Based on the above characterization, best emulsion was chosen for its bioassay against housefly adult (repellency assay) and larvae (larvicidal assay). The assay was done both with freshly prepared emulsion and with emulsion stored for 12 months at ambient temperature (varying between 10 ± 5 ◦ C and 40 ± 5 ◦ C). 2.6.1. Repellency assay The repellent effects of M. × piperita oil formulation was evaluated against field-collected adult houseflies (n = 50) in a repellency chamber (Kumar et al., 2011b). Repellency chamber consisted of an outer chamber (20 cm × 20 cm × 20 cm) which was connected to an inner chamber (60 cm × 60 cm × 60 cm) by a hole measuring 9 cm × 9 cm. Outer chamber contained a Petri plate (dia. – 9 cm) containing filter paper uniformly impregnated with test solutions of emulsion formulation. Three different dilutions of M. × piperita oil emulsion, 1, 3, and 5%, corresponding to the concentration of 0.003, 0.009 and 0.016 L/cm2 was used as test solution. In the control treatment, the paper was treated with a mixture of aromax and surfactant. The experiment was performed for 4 h, at the end of which the number of flies repelled from outer to inner chamber was counted to determine the percentage repellency. Three replicates of each treatment were performed. 2.6.2. Larvicidal bioassay Larvicidal bioassay was performed with lab reared housefly larvae. Larvae (20, 2nd instar) were placed on a filter paper containing a diet of 2 g groundnut oil cake, 5 g wheat bran, 2 g milk powder and 1 g honey mixed with 10 mL water. In a pour-on treatment, 500 L of M. × piperita oil emulsion at the dilutions of 1%, 3% and 5% in water, corresponding to oil concentration of 0.003, 0.009 and 0.016 L/cm2 was added to the diet. Diet material in the control treatment was sprayed with a mixture of aromax and surfactant. Three replicates of each oil treatment were performed. Larvae were observed for any change in appearance and mobility over the subsequent 48 h. Larval mortality was assessed by withering and the development of a brownish appearance (Kumar et al., 2011b). 2.7. Statistical analysis Mortality data were corrected for control mortality according to Abbott (1925) and normalized using arcsine transformation. Data for 50% and 90% lethal concentration for larval mortality (LC50 and LC90 ) were calculated using SPSS (2007) software. 3. Results and discussion 3.1. Effect of homogenization Effect of homogenization on emulsion was studied in relation to its outcome on relative creaming volume, particle size and zeta potential. Effect of homogenization on relative creaming volume showed decrease in creaming layer thickness with each homogenization cycle (Fig. 1). For each EC, creaming volume decreased by more than half with each homogenization cycle; with no creaming layer being observed in EC ‘B’, after 2nd homogenization
Fig. 1. Effect of homogenization on relative creaming volume of emulsified concentrate (EC).
cycle. Creaming volume has been used as stability criteria for orange oil emulsion and is reported to be influenced by surfactant concentration (Edris, 1998). Effect of homogenization on particle size of ECs is represented in Fig. 2. A slight decrease in average particle size with each homogenization cycles was observed for EC ‘B’ (Fig. 2B). Similarly, the particle size of EC ‘C’ after 1st, 2nd and 3rd homogenization cycles was 4207.8 nm, 3684 nm, and 1133.6 nm, respectively. Homogenization assists emulsion formation by providing mechanical energy, which helps in emulsion stability by counteracting particle coalescence (Chen and Tao, 2005). Hence, homogenization is expected to decrease the average particle size. However, the average particle size of EC ‘A’ showed variability with homogenization cycles; being 345.9 nm (after 1st homogenization cycle), 852.6 nm (after 2nd homogenization cycle) and 563.7 nm (after 3rd homogenization cycle) (Fig. 2A). The increase of droplet size reflected an ongoing coalescence process inside the emulsion, thus decrease in stability. The apparent disparity in particle size of emulsion with homogenization procedure is probably due to effect of molecular properties of surfactant, surfactant–solvent interaction and self-aggregated structure formed by surfactants molecules, which has significant role in determining particle size and polydispersity of emulsion droplets (Rodríguez-Abreu and Lazzari, 2008). In the present study, EC ‘B’ had equal concentration of both the surfactants, while the concentration of surfactants (CABS-70:NP-20) was more skewed for EC ‘A’ (0.7) and EC ‘C’ (0.5). The variation in surfactant concentration is reflected in particle size of emulsions, in the present study. Also, homogenization effect on emulsion stability is apparent, particularly for EC ‘C’. Roland et al. (2003) also reported variation in median size of the emulsion with surfactant concentration and homogenization cycle, while Varona et al. (2009) observed homogenization velocity to have greatest impact on emulsions’ particle size. Zeta potential measurement for different ECs with homogenization cycles are presented in Fig. 3. All the values of zeta potential were strongly negative and lied between −43 mV and −54 mV, reflecting emulsion stability. Zeta potential measures the repulsive force created by surfactant adsorption on the surface of dispersed phase droplet of emulsion, thus characterizes deflocculation capacity of emulsion and reflects its stability (Roland et al., 2003). Although, a general decrease in value of zeta potential was observed with each homogenization cycle, the changes were not as significant as in the case of creaming volume and particle size. The above results showed the significance of homogenization cycle on stability of emulsion with varied surfactant concentrations. Also, the optimization of surfactant concentration is crucial to produce a stable end product as reflected by values of creaming volume, particle size and zeta potential (Dickinson, 1992).
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Fig. 2. Particle size of emulsified concentrate (EC): (A) EC ‘A’, and (B) EC ‘B’, after (a) 1st, (b) 2nd, and (c) 3rd homogenization cycles.
Fig. 3. Zeta potential of emulsified concentrate (EC): (A) EC ‘A’, (B) EC ‘B’, and (C) EC ‘C’, after (a) 1st, (b) 2nd, and (c) 3rd homogenization cycles.
3.2. Characterization of EC formulations The properties of different emulsions prepared with varied concentrations of two surfactants; CABS-70 (anionic) and NP-20
(non-anionic) are shown in Table 1. All the ECs were reddish in appearance. ECs A and B exhibited slightly acidic pH while EC ‘C’ had slightly alkaline pH. Viscosity of EC ‘A’, ‘B’ and ‘C’ were 25, 28.5 and 30 cP, respectively. Viscosity of emulsion is proportional
Fig. 4. Effect of temperature on emulsified concentrate (EC ‘B’).
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Fig. 5. Effect of storage temperature on relative creaming volume of emulsified concentrates (ECs).
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Fig. 6. Effect of centrifugation on relative creaming volume of emulsified concentrates (ECs).
to volume of dispersed phase, and is also influenced by surfactant concentration (Tadros, 1994). The flash point is a measure of the tendency of a sample to form flammable mixtures with air in controlled laboratory conditions and is parameter for storage and handling consideration of flammable materials (Encinar et al., 2005). Flash point of ECs varied between 90 and 92 ◦ C, which was approximately equivalent to flash point of M. × piperita essential oil (92 ◦ C). The flash point of formulation obtained in this study, was much higher than the prescribed minimum limit of 24.5 ◦ C (Kumar and Parmar, 2000). Relatively high flash point of the product observed in this study, makes it less volatile and safer to transport and handle. Particle size of EC ‘B’ was observed to be lowest at 223.5 nm. After a storage period of 12 months, average particle size of emulsion showed an increment of 31–48%, probably due to agglomeration of emulsion droplets. Creaming volume measurement of stored emulsion also showed significant increase (Table 1).
stability of EC ‘B’ which showed relative creaming volume of 2%, after 3rd centrifugation cycle. Minimum stability was found in EC ‘C’, having relative creaming volume of 6%, after 3rd centrifugation cycle. The stability of ECs under centrifugation pressure reflects the strength of the interfacial film, thus stability of emulsion (Puisieux and Seiller, 1983). Freeze/thaw cycles showed no visible change in appearance in EC ‘A’ and ‘B’, while EC ‘C’ showed tendency to rupture, i.e. dispersion of oil droplets in emulsion, after 6 cycles. The result obtained in this study was better than Roland et al. (2003) who reported rupturing of the emulsions after five cycles. Similarly, in the study by Kumar and Parmar (2000) cold treatment of neem oil ECs at 0 ◦ C showed turbidity and slight solid separation. Overall, the various physical properties of ECs showed a correlation, with one property complimenting another. This could be comprehended for a particular EC (or emulsion), where value of all the three indicated properties (creaming volume, particle size and zeta potential) pointed to their stability. For EC ‘B’, the value of their creaming volume and particle size, both signified their better stability compared to EC ‘A’ or EC ‘C’. Results of various process variables also indicated EC ‘B’ as most stable to any external changes. The difference in stability for ECs could be attributed to the variation in surfactant concentration. As is evident from the results, EC containing equal ratio of surfactants, CABS-70:NP-20 (EC ‘B’) was most stable, while stability showed propensity to decrease with the skewing of surfactant ratio away from its mid point.
3.3. Effect of process parameters Storage of ECs at different temperature demonstrated yellowish-reddish-dark reddish appearance (Fig. 4). Relative creaming volumes of emulsions at various temperatures are shown in Fig. 5. Highest relative creaming volume was seen in EC ‘C’ at different temperatures, while EC ‘A’ and EC ‘B’ showed comparatively thin creaming layer. Emulsions were stable at the temperature of 4, 15, 30 ◦ C and to some extent even at 45 ◦ C. At the temperature of 60 ◦ C, phase separation in emulsion was observed, and thus was not considered suitable for emulsion stability during storage. The result obtained in this study was compatible with the one obtained by Edris (1998) who reported increase in creaming layer of orange oil emulsion with increase in temperature, and obtained lowest creaming volume at 4 ◦ C. Centrifugation is used to assess the stability of emulsion by speeding up potential destabilization process such as, creaming and sedimentation. Effect of centrifugation on emulsions is shown in Fig. 6. Emulsion showed thin creaming layer at the upper surface without any phase separation. The figure represents maximum
3.4. Bioassay against housefly On the basis of various characterizations EC ‘B’ was adjudged as best, being most stable and having lower sedimentation tendency. Thus, EC ‘B’ was chosen for bioassay study against housefly adults and larvae. Rates of repellency of adult houseflies by different concentration of M. × piperita oil emulsion are shown in Fig. 7. Emulsion prepared from freshly prepared EC showed 70–88% repellency at the end of 4 h of observation, which decreased to 34–72% of repellency for emulsion prepared from EC formulation stored for 12 months at
Table 1 Properties of three emulsified concentrate (EC) formulation of M. × piperita oil. Properties
A
B
C
pH Electrical conductivity (S/cm) Flash point (◦ C) Fire point (◦ C) Viscosity (cP) Particle size (nm) Zeta potential (mV) Particle size after 12 month storage (nm) Relative creaming volume (%) Relative creaming volume after 12 month storage (%)
6.6 88 92 102 25 536.7 −45.9 712.2 0.4 3.7
6.65 47.7 91 101 28.3 223.5 −43.8 293.9 0 2.1
7.54 30.8 90 101 30 1133.6 −47.3 1680.0 0.8 7.7
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Table 2 Lethal concentration (LC50 and LC90 ) and lethal time (LT50 ) of M. × piperita oil emulsions against 2nd instar larvae of Musca domestica. Mentha oil emulsion
Dilution (%)
Concentration (L/cm2 )
LT50 (h)
LC50
After 0 month
1 3 5
0.003 0.009 0.016
17.1 11.3 9.8
0.08
0.20
After 12 month
1 3 5
0.003 0.009 0.016
59.3 52.7 40.4
0.34
0.49
[12 h]
(L/cm2 )
LC90
[12 h]
(L/cm2 )
In their study, the crude form of essential oil showing higher efficacy (Thymus herba-barona essential oil), maintained its activity in formulation. The discussion from the above literature studies also opines the significance of oil and surfactant selection and preparation methodology on emulsion properties and also, on emulsion bioefficacy. In context of housefly control, emulsion of essential oil being superior spray carrier, could play significant role for the control of gregarious adult flies. Also, emulsion formulations are excellent for application against insects which tend to hide through opening or cervices, viz. stored grain pests, housefly larvae. However, in view of the absolute paucity of literature pertaining to essential oil emulsions for housefly control, the current report makes a pioneering contribution. Fig. 7. Repellency (%) of Musca domestica adults by M. × piperita oil emulsion at ) indicate emulsion from freshly prepared different concentration [solid lines ( emulsified concentrate; broken lines ( ) indicate emulsion from emulsified concentrate stored for a period of 12 months; 0.016 L/cm2 ; 0.009 L/cm2 ; 䊉 0.003 L/cm2 ].
the room temperature. Larvicidal bioassay for emulsion from both freshly prepared and stored EC showed significant variation with doses applied, time of observation and interaction between doses and time. Lethal concentration, LC50 for emulsion from freshly prepared EC was 0.08 L/cm2 while emulsion of stored EC showed LC50 value of 0.34 L/cm2 (Table 2). The results obtained from the present study indicates slight loss in efficacy of EC formulation over storage period, insecticidal properties of formulation was not lost completely, even after storage period of 12 months. Hence, enhanced dosage of the stored EC formulation would be required for comparable efficacy. The formulation was stored at room temperature, with ambient storage temperature fluctuating between 10 ± 5 ◦ C and 40 ± 5 ◦ C. Significant temperature variations could have lead to the loss in bioactivity. The depreciation in emulsion bioefficacy could also be attributed to reduction in its stability due to agglomeration of oil droplets over storage period as reflected by the particle size and creaming volume analysis (Table 1). However, more than 90% of the oil remained in the emulsified form for all the emulsions. Compared to the result of present study, lavandin essential oil has been reported to retain 80% of the oil in the stabilized form after 50 days of storage at 5 ◦ C (Varona et al., 2009). Therefore, the stability results obtained in the present study for Mentha oil emulsion, pertaining to storage for 1 year at ambient temperature, is quite encouraging. Further improvement in shelf life can be achieved by storage under controlled temperature conditions and the investigations on these lines are underway. As discussed earlier, only few studies could be retrieved on essential oil formulations for insect control. Kumar and Parmar (2000) prepared and evaluated neem oil based emulsions against 2nd instar larvae of hairy caterpillar, Spilosoma obliqua. In their study, they deduced the dependence of emulsion activity on surfactant types and concentration. In another study, Moretti et al. (2002) reported variation in emulsion bioefficacy against larvae of gypsy moth, Limantria dispar, with variation in essential oils.
4. Conclusions In an attempt to utilize biological control potential of M. × piperita oil, an emulsion formulation was prepared. Preparation methodology highlighted the variation in emulsion properties (creaming volume, particle size and zeta potential) with homogenization cycle and surfactant concentration. Characterization of emulsion for its pH, viscosity and flash point analysis indicated a stable formulation, which was safe to handle and transport. Further, stability of emulsion was established at various process parameters (temperature, centrifugation and free-thaw properties). Bioefficacy of emulsion against adults and larvae of housefly was found to be appreciable at various dilution, but showed slight decrease in efficacy with storage period. Acknowledgments The authors acknowledge Mr. Jai Kumar (Formulation Division, IPFT, Gurgaon) and Mr. Sabal Singh (IIT Delhi, India) for their help in experimental work. Partial financial support from KVIC Interface project is gratefully acknowledged. References Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46, 446–475. Bylaite, E., Nylander, T., Venskutonis, R., Jönsson, B., 2001. Emulsification of caraway essential oil in water by lecithin and b-lactoglobulin: emulsion stability and properties of the formed oil–aqueous interface. Colloids Surf. B: Biointerfaces 20, 327–340. Carrillo-Navas, H., Cruz-Olivares, J., Varela-Guerrero, V., Alamilla-Beltrán, L., VernonCarter, E.J., Pérez-Alonso, C., 2012. Rheological properties of a double emulsion nutraceutical system incorporating chia essential oil and ascorbic acid stabilized by carbohydrate polymer–protein blends. Carbohydr. Polym. 87, 1231–1235. Chen, G., Tao, D., 2005. An experimental study of stability of oil–water emulsion. Fuel Process. Technol. 86, 499–508. Dickinson, E., 1992. Interfacial interactions and the stability of oil-in water emulsions. Pure Appl. Chem. 64, 1721–1724. Edris, A.E., 1998. Preparation and stability of a protein stabilized orange oil-in-water emulsion. Nahrung 42, 19–22. ElShafei, G.M.S., El-Said, M.M., Attia, H.A.E., Mohammed, T.G.M., 2010. Environmentally friendly pesticides: essential oil-based w/o/w multiple emulsions for anti fungal formulations. Ind. Crops Prod. 31, 99–106.
P. Kumar et al. / Industrial Crops and Products 44 (2013) 611–617 Encinar, J.M., Juan, F., Gonzalez, J.F., Rodriguez-Reinares, A., 2005. Biodiesel from used frying oil: variables affecting the yields and characteristics of the biodiesel. Ind. Eng. Chem. Res. 44, 5491–5499. Kumar, L., Parmar, B.S., 2000. Effect of emulsion size and shelf life of azadirachtin A on the bioefficacy of neem (Azadirachta indica A. Juss) emulsifiable concentrates. J. Agric. Food Chem. 48, 3666–3672. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011a. Insecticidal properties of Mentha species: a review. Ind. Crops Prod. 34, 802–817. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011b. Repellent, larvicidal and pupicidal properties of essential oils and their formulations against the housefly, Musca domestica. Med. Vet. Entomol. 25, 302–310. Kumar, P., Mishra, S., Malik, A., Satya, S., 2012. Efficacy of Mentha × piperita and Mentha citrata essential oils against housefly, Musca domestica L. Ind. Crops Prod. 39, 106–112. Moretti, M.D.L., Sanna-Passino, G., Demontis, S., Bazzoni, E., 2002. Essential oil formulations useful as a new tool for insect pest control. AAPS Pharm. Sci. Tech. 3, 1–11.
617
Puisieux, F., Seiller, M., 1983. Galenica-Agents de surface et émulsions. Lavoisier, Paris. Rodríguez-Abreu, C., Lazzari, M., 2008. Emulsions with structured continuous phases. Curr. Opin. Colloid Interface Sci. 13, 198–205. Rodríguez-Rojo, S., Varona, S., Núnez, M., Cocero, M.J., 2012. Characterization of rosemary essential oil for biodegradable emulsions. Ind. Crops Prod. 37, 137– 140. Roland, I., Piel, G., Delattre, L., Evrard, B., 2003. Systematic characterization of oil-in-water emulsions for formulation design. Int. J. Pharm. 263, 85– 94. SPSS, 2007. Statistical product and service solution, system user’s guide, version 17.5. Tadros, T.F., 1994. Fundamental principles of emulsion rheology and their applications. Colloids Surf. A 91, 39–55. Varona, S., Martín, Á., Cocero, M.J., 2009. Formulation of a natural biocide based on lavandin essential oil by emulsification using modified starches. Chem. Eng. Proc. 48, 1121–1128.