Parasitol Res (2012) 110:1173–1178 DOI 10.1007/s00436-011-2610-2
ORIGINAL PAPER
Larvicidal activity of Copaifera sp. (Leguminosae) oleoresin microcapsules against Aedes aegypti (Diptera: Culicidae) larvae Luiz Alberto Kanis & Josiane Somariva Prophiro & Edna da Silva Vieira & Mariane Pires do Nascimento & Karine Modolon Zepon & Irene Clemes Kulkamp-Guerreiro & Onilda Santos da Silva
Received: 28 July 2011 / Accepted: 4 August 2011 / Published online: 18 August 2011 # Springer-Verlag 2011
Abstract Studies have demonstrated the potential of Copaifera sp. oleoresin to control Aedes aegypti proliferation. However, the low water solubility is a factor that limits its applicability. Thus, the micro- or nanoencapsulation could be an alternative to allow its use in larval breeding places. The purpose of this study was to evaluate if achievable lethal concentrations could be obtained from Copaifera sp. oleoresin incorporated into polymers (synthetic or natural) and, mainly, if it can be sustained in the residual activity compared to the pure oil when tested against the A. aegypti larvae. Microcapsules were prepared by the process of emulsification/precipitation using the L. A. Kanis (*) : E. da Silva Vieira : M. P. do Nascimento : K. M. Zepon Grupo de Pesquisa em Tecnologia Farmacêutica—TECFARMA, Universidade do Sul de Santa Catarina, Av. José Acácio Moreira 787, Dehon, 88704-900 Tubarão, SC, Brazil e-mail:
[email protected] J. S. Prophiro Grupo de Pesquisa em Imunoparasitologia—IMPAR, Universidade do Sul de Santa Catarina, Av. José Acácio Moreira 787, Dehon, 88704-900 Tubarão, SC, Brazil I. C. Kulkamp-Guerreiro Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, CEP 90050-170 Porto Alegre, RS, Brazil O. S. da Silva (*) ICBS, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, sala 206, CEP 90050-170 Porto Alegre, RS, Brazil e-mail:
[email protected]
polymers of cellulose acetate (CA) and poly(ethyleneco-methyl acrylate) (PEMA), yielding four types of microcapsules: MicPEMA1 and MicPEMA2, and MicCA1 and MicCA2. When using only Copaifera sp. oleoresin, the larvicidal activity was observed at concentrations of LC50 = 48 mg/L and LC99 =149 mg/L. For MicPEMA1, the LC50 and LC99 were 78 and 389 mg/L, respectively. Using MicPEMA2, the LC50 was 120 mg/L and LC99 >500 mg/L. For microcapsules MicCA1 and MicCA2, the LC50 and LC99 were 42, 164, 140, and 398 mg/L, respectively. For a dose of 150 mg/L of pure oleoresin, the residual activity remained above 20% for 10 days, while the dose of 400 mg/L remained above 40% for 21 days. The MicPEMA1 microcapsules showed a loss in residual activity up to the first day; however, it remained in activity above 40% for 17 days. The microcapsules of MicCA1 showed similar LC50 of pure oil with 150 mg/L.
Introduction In recent decades, many studies have been conducted with the aim of identifying natural origin substances, which can be used in the development of insecticides, especially for vectors of human and animal pathogens. The main reasons in the search for new alternatives to control these vectors are the frequent changes in the susceptibility of insects to synthetic chemical insecticides and the challenge to use biodegradable products with reduced environmental impact, maintaining the biological efficacy (Pavela 2008; Rahuman et al. 2008; Mendonça et al. 2005). Studies to date show that oils (essential or not) extracted from plants are those with the best results in this field and become potential candidates for the development of natural
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insecticides (Conti et al. 2010; Cheng et al. 2009; Evergetis et al. 2009; Shanmugasundaram et al. 2008; Silva et al. 2006, 2008; Cavalcanti et al. 2004). Among the various oils with insecticidal activity, the oleoresin extracted by tapping the trunk of the trees from several Copaifera species (known as copaiba and originated from the Amazon forest) exhibits excellent larvicidal activity against Aedes aegypti vector of different diseases like dengue and yellow fever virus (Prophiro et al. 2011; Geris et al. 2008; Silva et al. 2003, 2007). The mainly biochemical substances known to cause mortality in mosquito larvae are dipertenoids (Geris et al. 2008). However, one of the limiting factors for the development of insecticides using vegetable oils, such as that obtained from Copaifera sp., is that the components have low water solubility. This property required the use of organic solvents to solubilize them, and most of the solvents are hostile to the environment (Anjali et al. 2010; Elek et al. 2010). This property impedes the direct and uniform application of oil on the sites of mosquito larval growth because the larvae grow in water media where it is difficult to disperse the oily product. An alternative technology to enable the application of oily substances extracted from plants could be through its incorporation into micro- or nanoparticles (Jayaseelan et al. 2011; Santhoshkumar et al. 2011; Paula et al. 2010; Gonsalves et al. 2009). The use of micro- and nanoparticles is currently the main direction of investment in the pharmaceutical area throughout the world. Through these products, it is possible to improve the chemical stability of substances, delineate their action location, and prolong the activity time (Domb et al. 2007; Fattal et al. 2007; Warikoo and Kumar 2006; Hirech et al. 2003). In the case of preparation of insecticides using hydrophobic substances such as oils, the use of nano- and microparticles (1 nm–1,000 μm) reduces its volatility (Flores et al. 2011) and can facilitate the application and dispersion of the product on site. In addition, the predominant feeding mode of A. aegypti is collecting–filtering and shredding of particulate matter that ranges from colloid size to 50 μm (Forattinni 2002; Einsenberg et al. 2000; Merritt et al. 1992). The majority of larvae ingest higher proportions of fine particulate organic material suspension, and the Table 1 Formulations of four nanocapsules prepared with Copaifera sp. oleoresin
Formulation
MicPEMA1 MicPEMA2 MicCA1 MicCA2
larvae are not very discriminating in the types of food they ingest (Merritt et al. 1992). This way, the incorporation of actives in nano- or micropolymeric particles might drive the ingesting of larvicidal substances by larvae (Dahl 1988). The purpose of this study was to obtain Copaifera sp. oleoresin microcapsules using a synthetic polymer [poly (ethylene-co-methyl acrylate)] and cellulose derivative (cellulose acetate), and evaluate if its incorporation into micropolymeric particle formulations could provide better results than the natural oil comparing lethal concentration and residual activity when tested against the A. aegypti larvae.
Materials and methods Materials The oleoresin was extracted from Copaifera multijuga and Copaifera reticulata, and purchased from Cooperative Amazooncop (Altamira, Para, Brazil). The oil was used as received. Poly(ethylene-co-methyl acrylate) (PEMA), cellulose acetate (CA), and polyethylene glycol distearate (PEG-DE) were obtained from Aldrich Co. (USA). Polysorbate 80 and acetone were purchased from Synth Co. Production of the microcapsules using Copaifera sp. oleoresin The nanocapsules were obtained by interfacial polymer deposition using PEMA and CA as polymers, the active substance Copaifera sp. oleoresin as core and tensoactives (Table 1). The organic phase was poured into water under magnetic stirring. The acetone was removed under reduced pressure at 38±2°C. The microcapsule solutions were kept under dark conditions until evaluation of larvicidal activity. Characterization of Copaifera sp. oleoresin microcapsules The microcapsules were characterized in terms of particle size and polydispersion. The mean diameter over the
Organic phase
Water phase
Active (mg) Oil
Co-tensoactive (mg) PEG-DE
Polymer (mg) PEMA
CA
500 300 250 150
100 100 100 100
150 150 – –
– – 90 90
Acetone (mL)
Tensoactive (mg) Polysorbate 80
Water (mL)
27 27 27 27
70 70 70 70
53 53 53 53
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volume distribution (d4.3) and particle size distribution (SPAN) (Eq. 1) were determined by laser diffractometry (Mastersizer 2000; Malvern Instruments, UK), where d0.9, d0.1, and d0.5 are the particle diameters determined at 90%, 10%, and 50% cumulative undersized volumes, respectively. SPAN ¼
d0:9 d0:1 d0:5
ð1Þ
Copaifera sp. oleoresin encapsulation efficiency (EE) The encapsulation efficiency of the Copaifera sp. oleoresin in the microcapsules was determined by using UV–Vis spectrophotometer immediately after preparation. The total Copaifera sp oleoresin contents (TC) of the samples were calculated from a standard curve prepared with Copaifera sp. oleoresin dilutions measured by a spectrophotometer (Bell, LGS53) at 252 nm (maximum UV absorbance determined from a scanning between 210 nm and 600 nm). Non-encapsulated Copaifera sp. oleoresin (free oleoresin) (FC) was determined by the addition of 2 mL of microcapsules to 6 mL of ethyl acetate and was vortexed for 3 min, followed by centrifugation for 5 min at 4,000 rpm; the supernatant was collected for UV analysis. The encapsulation efficiency (EE) was determined by comparing the actual quantity of oleoresin incorporated with the quantity of the initial oleoresin incorporated to formulation (Eq. 2) (Surassmo et al. 2010). TC FC EEð%Þ ¼ 100 ð2Þ TC Mosquitoes Rockefeller strains of A. aegypti are continuously maintained in laboratory, being reared under a 14-h light/10-h dark photoperiod. Larvae were reared on powdered puppy food (Purina® Cat Chow®), 0.2 g/100 mL, three times a week. Adult males and females were continuously provided with a 5% honey solution, while females were blood fed on BALB/c mice twice a week in order to obtain eggs for colony development. All bioassays were determined under 25°C and 80% (±10%) relative humidity in an ELETROlab® 132FC incubator. Lethal concentration determination The Copaifera sp. oleoresin solution at 700 mg/L was obtained by oil solubilization in 2% dimethyl sulfoxide (DMSO) in water at 40°C. This solution was used to obtain dilutions ranging from 10 mg/L to 160 mg/L in water. For MicPEMA and MicCA microcapsules, dilutions were
prepared at a range between 10 mg/L and 500 mg/L of microcoated Copaifera sp. oleoresin, in water. 100 ml of the solutions containing the Copaifera sp. oleoresin and microcapsules at different concentrations were placed in plastic containers. Then, 20 active larvae (late third or early fourth stage) were transferred to the solution. The bioassays were repeated three times for each concentration and larval stage. For each test, a control was used with water and 2% DMSO for Copaifera sp. oleoresin. For tests using microcapsules, the control was without Copaifera sp. oleoresin. The larval mortality was measured after 24 h of exposure to the solutions and confirmed when the larvae did not show any movement in response to being touched with entomological needles. The lethal concentrations (LC50 and LC99) were interpolated by the Probit analysis using GraphPad Prism computer program (GraphPad Software 1995, San Diego, CA, USA) and were reported as geometric means ± SD. Residual effect The experiments were performed using 150 and 400 mg/L of pure Copaifera sp. oleoresin previously dissolved with DMSO to verify the residual effect, and for Copaifera sp. oleoresin microcapsule dilutions of MicPEMA and MicCA were used containing proportional concentration of 400 mg/L and 150 mg/L of oil resin. Four replicates were made, each containing 500 mL of solution in plastic containers with a capacity of 1,000 mL. 100% active late third or early fourth stage larvae were placed in each replica, with a total of 400 larvae per bioassay. Mortality was checked after 24 h of exposure to pure oil and microcapsules. Dying larvae, unable to reach the water surface when touched, were considered dead (WHO 1981a, b). The survived larvae were killed and then discarded. Then, the solution of each replica was filtered and 100 new healthy larvae were placed in the solution. This method was repeated until no more larval mortality was observed in the solutions containing oil. The same procedure was performed with the control groups.
Results Mean diameters, size distribution (SPAN), and rate oil/ polymer of microcapsules prepared, as so the encapsulation efficiency of oleoresin, are shown in Table 2. The particles presented mean diameters in the range from 12.1 μm to 57.8 μm as expected for microparticles. Also, since the SPAN is the measurement of the width of the distribution, we observed lesser size distribution and better encapsulation efficiencies for microcapsules prepared with CA polymer. This could be related to the characteristics of polymer used, as so influenced by the minor concentration of
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Table 2 Mean diameters, span size distribution, rate oil/polymer, and encapsulation efficiency (EE) of microcapsules formulation Formulation
Span
Mean diameters (d4.3) (μm)
Oil/polymer rate
EE (%)
MicPEMA1 MicPEMA2 MicCA1 MicCA2
134.2 7.5 2.2 2.7
12.1 57.8 21.9 19.6
3.3 2.0 2.8 1.7
75.7 88.2 98.5 93.0
polymer in these formulations. On the other side, the formulation containing both the highest polymer and oil concentrations showed the largest size distribution. The suspensions containing dispersions of the microcapsules of PEMA and CA showed no oil phase separation during 60 days of study. On the other hand, the dilutions of pure Copaifera sp. oleoresin using an organic cosolvent DMSO showed the presence of a thin layer of oil on the surface of the solution after 3 days of preparation, indicative of phase separation. Both suspensions of MicPEMA and MicCA were easily dispersed in water without any additives, when used for larvicidal studies. However, the Copaifera sp. oleoresin required pre-solubilization using DMSO at 40°C. Table 3 presents the values of LC50 and LC99 for Copaifera sp. oleoresin and microcapsules containing such oil. As it can be seen, LC50 obtained in the tests by using MicPEMA1 and MicPEMA2 showed an increase of 1.6 and 2.5 times, respectively, when compared to pure Copaifera sp. oleoresin. It was not possible to determine the LC99 for MicPEMA2 with the dilutions used in the experiment because the value was above 500 mg/L. For a p 500 164 (142–171) 398 (372–421)
LC50 ,and LC99: lethal concentration that causes 50% and 99% mortality, respectively; CI 95%: % CI 95% confidence level
Fig. 1 Residual activity of Copaifera sp. oleoresin solutions at 150 mg/L (filled up-pointing triangles) and 400 mg/L (filled circles), MicPEMA1 at 400 mg/L (filled squares), and MicCA1 at 150 mg/L (filled down-pointing triangles)
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water, and ability to form a layer under water surface. The microencapsulation of Copaifera sp. oleoresin facilitated the dispersion into water when tested on larvae of A. aegypti, explained by the formulation composition and the microcapsule size (1–50 μm). The polymeric microparticles with low diameter size and low density produce buoyant or semi-buoyant particles (Hirech et al. 2003). It can increase the suspension time of particles in the environment and also increase the time required to possible capture by the larvae (Dahl 1988). Although this objective was achieved, the MicPEMA1, MicPEMA2, and MicCA2 microcapsules needed an increase in the oil concentration or a higher liberation to obtain the same mortality when compared to pure oleoresin. This result can be explained by the encapsulation of oleoresin, a fact that would delay the release of oil through the environment, consequently reducing its activity. The effect of altering the ratio of oil/polymer on the lethal doses is an indication that the polymer encapsulation affects larvicidal activity. The microcapsules obtained from PEMA in the ratio of oil to polymer equal to 2 had higher LC50 and LC99 >500 mg/mL, increasing the rate oil/ polymer to 3.3 as the LC is decreasing. The EMA polymer forms a dense film and free from porosity (Kanis et al. 2007), which can reduce the oil release and explain the decrease in larvicidal activity. For microcapsules produced with CA in the ratio of oil/ polymer 2.8 (MicCA1), the results were different from those observed in MicCA2, MicPEMA1, and MicPEMA2 since the lethal dose did not differ from pure oleoresin. This result can be explained by the lower proportion of polymer in relation to the amount of oil. It also gives CA films highly porous properties especially when it is formed in the presence of water (Meier et al. 2004). This porosity promotes a rapid release of oleoresin into the environment favoring the larvicidal activity. The time of maximum residual activity of pure oleoresin of Copaifera sp. is associated with the concentration used, and this proved to be superior to the microcapsules. In solutions containing Copaifera sp. oleoresin at a concentration of 150 and 400 mg/L, the formation of a thin layer of oil on the surface of solutions containing the larvae was observed during the study. This is a fact attributed to the physical instability of the solution, and that may have facilitated the ingestion of oleoresin by the larvae during the breathing process. The presence of an oleoresin film on the surface is a factor that can affect not only the development of larvae but also the breathing from other aquatic organisms, which is of great importance to the balance of the environment. The residual activity of microcapsules using Copaifera sp. oleoresin was determined only for formulations which had lower LC99 (MicPEMA1 and MicCA1). Residual
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activity showed a similar activity of MicCA1 and Copaifera sp. oleoresin at a concentration of 150 mg/L, explained by the properties of CA. Unlike the use of pure oil, the thin layer of oleoresin on the solution surface was not observed, indicating that the process of microencapsulation can promote a homogeneous dispersion and release of the oil in the aqueous environment. By comparing the residual activity of Copaifera sp. oleoresin solution at a concentration of 400 mg/L with MicPEMA1 dilutions containing proportionately the same oleoresin concentration, we observed that the residual activity was much lower for the microcapsules. This indicates that the oil incorporated in microcapsules is being released slowly and in small amounts to the larval environment. The extended release is expected for most of the microcapsules, and that could require a higher concentration of active substance applied to the means to ensure 100% of residual activity and supply the greatest uptime. However, we note that the total MicPEMA1 time on residual activity was lower than that for pure oil at the same concentration, indicating that part of the nanocoated oil got trapped in the polymer matrix of the microcapsule.
Conclusion The results showed that the microencapsulation of Copaifera sp. oleoresin with CA and PEMA for application as larvicide favored the dispersion of oleoresin in the solution and prevented the phase separation after a few days of application, different from those observed for the pure oleoresin. The PEMA does not seem to be a good alternative for Copaifera sp. oleoresin microencapsulation. Besides being a non-biodegradable synthetic polymer, it delayed the release of active oil. Furthermore, it did not increase the maximum time of activity. The CA, a polymer of natural origin, showed greater potential for obtaining microcapsules. With a reduced concentration of polymer, the microcapsules have the same effect as the oleoresin of Copaifera sp. Increasing the polymer concentration may be an alternative to extend the residual effect. Acknowledgments The authors thank the National Council of Research and Development (CNPq/410579/2006-8) and UNISUL for scholarships and financial support, and S.S. Guterres from Universidade Federal do Rio Grande do Sul for the particle size analysis.
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