Development of an insecticidal nanoemulsion with Manilkara subsericea (Sapotaceae) extract
- Caio Pinho Fernandes1, 2Email author,
- Fernanda Borges de Almeida1,
- Amanda Nunes Silveira3,
- Marcelo Salabert Gonzalez4,
- Cicero Brasileiro Mello4,
- Denise Feder4,
- Raul Apolinário4,
- Marcelo Guerra Santos5,
- José Carlos Tavares Carvalho6,
- Luis Armando Cândido Tietbohl7,
- Leandro Rocha7 and
- Deborah Quintanilha Falcão3
© Fernandes et al.; licensee BioMed Central Ltd. 2014
Received: 18 March 2014
Accepted: 9 May 2014
Published: 18 May 2014
Plants have been recognized as a good source of insecticidal agents, since they are able to produce their own defensives to insect attack. Moreover, there is a growing concern worldwide to develop pesticides with low impact to environment and non-target organisms. Hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea and its triterpenes were considered active against a cotton pest (Dysdercus peruvianus). Several natural products with insecticidal activity have poor water solubility, including triterpenes, and nanotechnology has emerged as a good alternative to solve this main problem. On this context, the aim of the present study was to develop an insecticidal nanoemulsion containing apolar fraction from fruits of Manilkara subsericea.
It was obtained a formulation constituted by 5% of oil (octyldodecyl myristate), 5% of surfactants (sorbitan monooleate/polysorbate 80), 5% of apolar fraction from M. subsericea and 85% of water. Analysis of mean droplet diameter (155.2 ± 3.8 nm) confirmed this formulation as a nanoemulsion. It was able to induce mortality in D. peruvianus. It was observed no effect against acetylcholinesterase or mortality in mice induced by the formulation, suggesting the safety of this nanoemulsion for non-target organisms.
The present study suggests that the obtained O/A nanoemulsion may be useful to enhance water solubility of poor water soluble natural products with insecticidal activity, including the hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea.
Chemical pesticides have been used to control pest insects, however, they are usually toxic to environment. There is a growing concern worldwide regarding indiscriminate use of these substances, which are associated to environmental pollution and toxicity risk to non-targeted organisms . Plant species are well recognized by their ability to produce defensive substances, in order to protect themselves from insect attack . These natural products appear as potential sources of new biodegradable insecticides with wide range of mechanisms of action, being an important alternative for insect pest management in agriculture . One of the most promising and recognized group of substances with insecticidal activity are the triterpenes .
Manilkara subscericea (Mart.) Dubard (Sapotaceae) is an endemic species of Brazilian Atlantic Forest  and widely distributed at Restinga de Jurubatiba National Park (Rio de Janeiro State, Brazil) . Several non-polar pentacyclic triterpenes have been described as major constituents of M. subsericea, mainly alpha- and beta-amyrin esters [7, 8]. Hexane-soluble fraction from ethanolic crude extract from fruits of M. subsericea and its major substances (alpha- and beta-amyrin acetate) was able to induce mortality, delayed development and inhibition of moulting in Dysdercus peruvianus , a hemiptera species which causes serious loss of cotton crops . This apolar fraction and its triterpenes have poor water solubility and are soluble in toxic organic solvents, such as chloroform and dichloromethane, being this intrinsic characteristic a technological challenge if development if a viable product is desired.
Nanotechnology has emerged as a promising area for development of products in a wide range of applications, including pesticide agents. Considering that many of the insecticides known today are organic compounds with poor water solubility, development of nanoproducts appear to solve this main problem, enhancing water solubility, bioavailability and resulting in stable formulations without utilization of organic toxic solvents . Nanoemulsions are one of the most important formulations to enhance solubility and dissolution properties of poorly water soluble substances . They are also referred as miniemulsions or ultrafine emulsions and have small droplet size (20-200 nm). They are transparent or translucent, often presenting a bluish reflect and have high kinetic stability . Low energy methods have been used to achieve nanoemulsions, including reverse-phase composition (RPC) and temperature of inversion phase (TIF) . Formulation screening stage is crucial if development of a stable nanoformulation is desired, especially if a low energy method is employed, being determination of required HLB value of an oil  and construction of pseudo-ternary phase diagrams  very useful, especially to achieve nanoemulsions.
On this context, the aim of the present study was to develop an insecticidal nanoemulsion containing apolar fraction from fruits of Manilkara subsericea and verify its effects against Dysdercus peruvianus and non-target organisms.
Results and discussion
Preliminary solubility studies were performed regarding choice of oil phase and surfactants. Octyldodecyl myristate (MOD®) was the best oil, being able to solubilize equal amount (1:1, w/w) of hexane-soluble fraction from fruits of M. subsericea (HF). It is frequently necessary to use blends, such as a pair of hydrophilic and lipophilic non-ionic surfactants, to achieve droplets with small diameter . Sorbitan oleate and polysorbate 80 were considered the best pair (Data not shown). These surfactants have been used in low energy methods, being able to produce nanoemulsions with smaller mean droplet size, when compared to other surfactants. This could be explained by the ability of this couple to induce formation of a looser film, which is associated to generation of nanoemulsions . Addition of water to a surfactant in oil solution was employed in the present study, since it provided better results, when compared to addition of oil to an aqueous surfactant solution (Data not shown). This could be attributed to phase transitions and changes in the curvature of the surfactant from W/O to O/W during emulsification process .
In order to predict the best ratio of surfactants to be used, several emulsions were prepared varying the relative amounts of sorbitan oleate and polysorbate 80. Most of them presented instable behavior, including critical macroscopical changes, such as creaming and phase separation. Surfactants can be classified according to their Hydrophile-Lipophile Balance (HLB), a semi-empirical scale  and several HLB values can be obtained using different amounts of each component of a couple of surfactants . Emulsions with HLB values of 10 (sorbitan oleate/polysorbate ratio, 1.0/1.1) and 11 (sorbitan oleate/polysorbate ratio, 1.0/1.7) were considered more stable. A second set of emulsions within this HLB range was prepared and the obtained formulations presented translucent aspect and bluish reflect, which is characteristic for nanoemulsions . Mean droplet size analysis indicated that nanoemulsion with HLB value of 10.75 (sorbitan oleate/polysorbate ratio, 1.0/1.5) presented the smallest mean diameter (50.6 ± 0.4 nm) and low polydispersity (0.164 ± 0.021). Stable formulations with low mean droplet size can be obtained when HLB value of the surfactant couple coincides with required HLB value of the oil [12, 20]. Thus, required HLB of oil can be determined by calculating the HLB value of emulsifier or emulsifier mixture which was able to induce formation of the most stable formulation, among a set of emulsions prepared with different blends of a couple of emulsifiers in a wide range of HLB value . Our results indicate that 10.75 should be the required HLB value of MOD® used in the present study.
Composition, mean droplet size and polydispersity of each formulation prepared during construction of pseudo-ternary phase diagram for delimitation of nanoemulsion region
% of oil
% of surfactants
% of water
Mean diameter (nm)
50.6 ± 0.4
0.164 ± 0.021
234.2 ± 12.5
0.025 ± 0.012
421.5 ± 50.4
0.005 ± 0.000
196.4 ± 12.5
0.178 ± 0.044
145.7 ± 8.6
0.132 ± 0.038
256.9 ± 8.6
0.016 ± 0.011
151.7 ± 6.0
0.155 ± 0.018
139.4 ± 9.6
0.078 ± 0.049
133.5 ± 0.5
0.247 ± 0.011
139.5 ± 4.7
0.073 ± 0.039
86.8 ± 0.9
0.294 ± 0.006
48.7 ± 0.2
0.313 ± 0.002
234.4 ± 2.3
0.270 ± 0.016
298.5 ± 31.8
0.005 ± 0.000
85.4 ± 1.2
0.350 ± 0.005
51.7 ± 0.2
0.331 ± 0.005
162.4 ± 1.3
0.335 ± 0.011
159.2 ± 2.3
0.334 ± 0.007
68.7 ± 0.8
0.360 ± 0.004
87.9 ± 1.5
0.365 ± 0.003
170.6 ± 6.2
0.144 ± 0.027
66.6 ± 1.1
0.304 ± 0.005
120.1 ± 1.0
0.225 ± 0.003
95.3 ± 0.6
0.255 ± 0.006
45.9 ± 0.4
0.271 ± 0.005
75.4 ± 2.3
0.340 ± 0.005
161.6 ± 1.9
0.266 ± 0.007
97.2 ± 1.0
0.256 ± 0.002
Special attention was given to formulation comprised by 5% of MOD, 5% of surfactants and 90% of water, which also presented stable behavior, small mean droplet size (50.6 ± 0.4 nm) and low polidispersity (0.164 ± 0.021). Low surfactant percentage could be considered an advantage, since further preparation of this formulation would reduce toxicity and costs with raw materials, when compared to other nanoemulsions with higher concentrations of surfactants. Thus, this formulation was chosen to prepare a nanoemulsion with hexane-soluble fraction from fruits of Manilkara subsericea dispersed through internal phase (HFNE).
HFNE and blank nanoemulsion presented zeta potential values of – 47.4 ± 3.2 and – 59.6 ± 4.1, respectively. Zeta potential is a special parameter that should be analyzed, in order to determine stability of nanoemulsions and is associated to surface potential of the droplets . Maximum stability is observed when zeta potential value is above ± 30 mV . The high stability of formulations with great zeta potential values is associated to repulsive forces that exceed attracting Van der Waals forces, resulting in dispersed particles and a deflocculated system . Macroscopical analysis of the nanoemulsion with HF and blank nanoemulsion indicated that these formulations maintained their original fine appearance and bluish reflection. It was observed no phase separation, creaming and sedimentation under room temperature (25 ± 2°C) and accelerated stability evaluation. Long term physical stability of a nanoemulsion related to its small droplets, making this type of formulation being also referred as “approaching thermodynamic stability” [26, 13].
Changes in the time period in which occur the processes of molt and metamorphosis were observed in group treated with HFNE and negative group (Data not shown). Moreover, an associated high mortality rate were displayed continuously and gradually increasing throughout insects lifecycle regardless whether the insects were in the nymphal or adult stage. This observation point out to a physiological connection between the neuroendocrine control of the insect development and the reduced longevity obtained after treatments. These results suggest that HFNE may able to release insecticidal components from HF, while formulation used as blank nanoemulsion may be used to disperse other insecticidal agents.
Weight variation in adult female and male Swiss albino mice ( Mus musculus ) treated with HFNE (5% of MOD®, 5% of surfactants (HLB of 10.75), 5% of hexane-soluble fraction from fruits of M. subsericea and 85% of water) by oral route, corresponding to 3 g/kg of extract
Body weight (Male)
Body weight (Female)
49.06 ± 0.43
50.39 ± 1.37
52.72 ± 1.62
50.64 ± 0.63
50.65 ± 1.50
52.05 ± 2.71
50.64 ± 0.63
51.76 ± 1.59
Previous study performed by our research group indicated that hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea presented insecticidal activity against Dysdercus peruvianus. This activity may be partially attributed to beta-and alpha amyrin acetates, which may be used as chemical markers for quality control of products with M. subsericea extracts. However, these substances, as well as the active fraction are poorly water soluble. As part of our ongoing studies with this species, we decided to develop an insecticidal nanoemulsion. This formulation was able to induce mortality in insects and our results suggest that it may be safe for non-target organisms and environment. The present study suggests the obtained O/A nanoemulsion may be useful to enhance water solubility of poor water soluble natural products with insecticidal activity, including the hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea. The absence of organic toxic solvents and stability makes this nanoemulsion a potential insecticidal product.
Materials and methods
Sorbitan oleate (HLB: 4.3) and Polysorbate 80 (HLB: 15) were purchased from La Belle Ativos Ltda (Paraná, Brazil). Octyldodecyl myristate (MOD®) was purchase from Brasquim Ltda (São Paulo, Brazil). Acetylthiocholine iodide (ATCI), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), physostigmine (eserine) and acetylcholinesterase from electric eel (type VI-S, C3389-2UK, lyophilized powder) were purchased from Sigma (Sigma-Aldrich Corporation, St Louis, MO). Hexane-soluble fraction from fruits of M. subsericea was previously obtained  and stored at 4°C for further utilization.
Emulsions were prepared by temperature of inversion phase method . The required amounts of both emulsifiers were dissolved in the oil phase and heated at 75 ± 5°C, while the aqueous phase was separately heated at same temperature. When both phases reached the same temperature, aqueous phase was gently added and mixed with the oil phase, using a mechanic agitator model Fisatom 713D at 400 rpm for 10 min and additional 5 min of agitation under cooling. Aditional constituents was weight an placed together with oil and surfactants mixture, being its mass discounted from water mass.
Required HLB determination
Each emulsion was prepared at a final mass of 25 g, containing 90% (w/w) of distilled water, 5% (w/w) of MOD® and 5% of a mixture of emulsifiers . Series of emulsions were prepared using sorbitan oleate (HLB = 4.3) and polysorbate 80 (HLB = 15), allowing a wide range of HLB values from 4.3 (5% w/w of sorbitan oleate) to 15 (5% w/w of polysorbate 80) by blending together the emulsifiers in different ratios.
Pseudo-ternary phase diagram
Nanoemulsion region was determined using pseudo-ternary phase diagram. Each corner corresponded to 100% of water, surfactants and MOD®. Surfactants blend was kept constant and corresponded to ratio which results on required HLB value of oil phase. Composition (w/w) which allowed required HLB value determination was used as starting point (90% of distilled water, 5% of oil and 5% of surfactants blend) and mean droplet size of each prepared composition was performed in order to determine nanoemulsion region.
Stability of all emulsions was evaluated immediately and after 1, 15 and 30 days of manipulation by macroscopic analysis, such as color, visual aspect, phase separation, creaming and sedimentation. During this period all emulsions were maintained under room temperature (25 ± 2°C) in screw-capped glass test tubes . Acelerated stability evaluation was performed keeping emulsion under controlled temperature (40 ± 5°C).
Droplet size and zeta potential analysis
The droplet size, polydispersity and zeta potential were determined by photon correlation spectroscopy using a ZetaPlus (Brookhaven Inst. Corp., USA). Each emulsion was diluted using ultra-pure Milli-Q water (1:25). Measurements were performed in quintuplicate and average droplet size was expressed as the mean diameter.
Dysdercus peruvianus were obtained from the colony maintained in the Laboratory of Insect Biology of the Universidade Federal Fluminense (GBG-UFF), being kept at 24-25°C, relative humidity of 70-75% and a 16:8 h light:dark cycle .
Fourth-instar insects were randomly chosen and separated in two treated groups, being one group topically applied with a nanoemulsion containing hexane-soluble fraction from fruits of M. subsericea (HFNE) (5% of MOD®, 5% of surfactants (HLB of 10.75), 5% of HF and 85% of water), corresponding to 50 μg of extract per insect, while negative control group was treated with blank nanoemulsion (5% of MOD®, 5% of surfactants and 90% of water). Untreated insects received no treatment, being only fed. Biological evaluation was performed in order to determine mortality levels during the entire time required for development from the fourth instar to the adult stage [9, 33, 34]. All experiments were repeated at least three times with samples from 30 insects (n = 30 in each triplicate). Significance of the results was analysed using ANOVA and Tukey’s test21 according to Stats Direct Statistical Software, v.2.2.7 for Windows 98. Differences between treated group and control.
Anticholinesterase activity was performed according to method described by Ellman et al. (1961)  with some modifications , using a 96-well microplate. A total volume of 200 μL of test media was composed by 65 μL of Phosphate buffered saline (PBS), 60 μL of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) 1,5 mM, 25 μL of electric eel acetylcholinesterase (Sigma) (AchE) 550 mU/mL, 25 μL of nanoemulsion and 25 μL of acetylthiocholine iodide (ASCh). Different concentrations of HF and eserine (positive control) were obtained by dilution of each nanoemulsion with PBS. Negative control was performed using a blank nanoemulsion, without inhibitor or extract. The spontaneous hydrolysis of substrate was calculated replacing the enzyme solution by PBS. Absorbance was measured at 412 nm. The statistical analysis of the anticholinesterase assay was performed on GraphPad Prism 5.04 program using Pearson’s correlation coefficient with 95% confidence interval.
This study was approved by the Ethics Committee of the Universidade Federal do Amapá (CEP – UNIFAP – 005AP/2013). All procedures were performed according to the International Committee for animal care in accordance with established national regulations for animal experimentation. The experiments were performed using adult female and male Swiss albino mice (Mus musculus), 12 weeks age, provided by the Central Laboratory of the State of Amapá – Macapá (LACEN/AP). Each experimental group was composed of 5 animals. They were kept in polyethylene cages on a temperature-controlled rack (25°C ± 2°C) under a 12-hour light-dark cycle. They had free access to food and water, except for the 24 hours before the experiments, when they had access only to water.
Acute toxicity studies were performed using both sexes of mice according to Pina et al. (2012) , with some modifications. Treated groups received a single dose of HFNE (5% of MOD®, 5% of surfactants (HLB of 10.75), 5% of hexane-soluble fraction from fruits of M. subsericea and 85% of water) by oral route, corresponding to 3 g/kg of extract. Negative control groups received a blank nanoemulsion (5% of MOD®, 5% of surfactants and 90% of water).
Observations were performed at 30, 60, 120, 240, 360 and 720 min after the oral treatment and daily for fourteen days. Behavioral changes (agitation, convulsions, vocal fremitus, irritation, stereotyped movements, touch response, salivation, tremors, writhing, body distension, ptose, sleepiness, defecation, diarrhea, piloerection), weight, food and water intake, clinical signs of toxicity and mortality were recorded daily. At the end of fourteen days, they were sacrificed by cervical dislocation and taken to autopsy for macroscopic observation of the organs (heart, lung, liver, kidney and spleen). Statistical analysis was performed by Student t test with 95% confidence intererval, using GraphPad Prism 5.04. Differences between organs, body weight and food and water intake were considered significant when p < 0.05.
Caio Pinho Fernandes is a professor at Universidade Federal do Amapá and has been working with natural products, including phytochemistry, nanotechnology and biological activities of these compounds.
Fernanda Borges de Almeida is an undergraduate student at Universidade Federal do Amapá and participated in this project as part of her scientific initiaion program.
Amanda Nunes Silveira is an undergraduate student at Universidade Federal Fluminense and participated in this Project as part of her scientific initiaion program.
Marcelo Salabert Gonzalez is professor at Universidade Federal Fluminense and has been working with complementary strategies to control insects with secondary metabolites from plant species.
Cicero Brasileiro Mello is professor at Universidade Federal Fluminense and has been working with complementary strategies to control insects with secondary metabolites from plant species.
Denise Feder is professor at Universidade Federal Fluminense and has been working with complementary strategies to control insects with secondary metabolites from plant species.
Raul Apolinário is undergraduate student at Universidade Federal Fluminense and participated in this project as part of her scientific initiation program and did al experiments with insects.
Marcelo Guerra Santos is professor at Universidade Estadual do Rio de Janeiro. He is a botanist and has been working with species from sandbanks of Parque Nacional da Restinga de Jurubatiba (RJ) Brazil.
José Carlos Tavares Carvalho is professor and President of the Universidade Federal do Amapá (Brazil) and has been working with natural products pharmacology.
Luis Armando Cândido Tietbohl is a Master’s student at Universidade Federal Fluminense and has been working with acetylcholinesterase inhibition.
Leandro Rocha is professor at Universidade Federal Fluminense and has been working with natural products and its biological activities.
Deborah Quintanilha Falcão is professor at Universidade Federal Fluminense and has been working with nanotechnology of natural products.
Authors would like to thank CAPES (n° 3292/2013 AUXPE), CNPQ and FAPERJ for the finantial support and “Centro Brasileiro de Pesquisas Físicas” for the use of Zeta Potential Analyzer.
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