Formation and stability of oil-in-water nanoemulsions containing rice bran oil: in vitro and in vivo assessments
© Bernardi et al; licensee BioMed Central Ltd. 2011
Received: 11 April 2011
Accepted: 28 September 2011
Published: 28 September 2011
Nanoemulsions have practical application in a multitude of commercial areas, such as the chemical, pharmaceutical and cosmetic industries. Cosmetic industries use rice bran oil in sunscreen formulations, anti ageing products and in treatments for skin diseases. The aim of this study was to create rice bran oil nanoemulsions using low energy emulsification methods and to evaluate their physical stability, irritation potential and moisturising activity on volunteers with normal and diseased skin types.
The nanoemulsion developed by this phase diagram method was composed of 10% rice bran oil, 10% surfactants sorbitan oleate/PEG-30 castor oil, 0.05% antioxidant and 0.50% preservatives formulated in distilled water. The nanoemulsion was stable over the time course of this study. In vitro assays showed that this formulation has a low irritation potential, and when applied to human skin during in vivo studies, the nanoemulsion improved the skin's moisture and maintained normal skin pH values.
The results of irritation potential studies and in vivo assessments indicate that this nanoemulsion has potential to be a useful tool to treat skin diseases, such as atopic dermatitis and psoriasis.
Where ω is defined as the rate of change of the cube of the number average radius, D is the diffusion coefficient of the dispersed oil phase in the aqueous phase, γ is the interfacial tension between the two phases, c∞ is the bulk solubility of the oil in the water and ρ is the oil density. k is a constant that has the value of 8/9 in the LSW.
Nanoemulsions are well characterised and are a promising drug delivery system with practical applications for pharmaceutical, cosmetic and chemical industry applications. They have been used in intravenous, oral and ocular drug administrations and have reduced drug side effects and improved the pharmacological effects of the drugs given [8–10, 4]. Nanoemulsions are primarily produced either by high-energy emulsification (e.g., high-pressure homogenisation) or by low-energy emulsification (using physicochemical properties of the components) . This work focuses on the latter method for nanoemulsion synthesis.
Rice (Oryza sativa) bran oil has unsaponifiable fractions that contain high levels of antioxidant-rich components, such as tocopherols/tocotrienols and gamma-oryzanol, which could be useful for topical formulations . The cosmetics industry has used rice bran oil in sunscreen formulations , in topical aging prevention products  and in treatments for skin diseases .
When skin is affected by diseases such as atopic dermatitis and psoriasis, it exhibits a compromised skin barrier function that causes increased transepidermal water loss [16–18]. Moisturisers can help improve the skin's function by relieving the cutaneous manifestations of these diseases [18–20]. Measuring the stratum corneum (SC) hydration degree gives important information about the biophysical properties and the function of the skin .
In vitro studies, such as the HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane), are used to evaluate cosmetics products by immediately showing whether or not a solid or liquid substance irritates the hen's egg chorioallantoic membrane .
The aim of this study was to obtain nanoemulsions of rice bran oil and to evaluate their physical stability, irritating potential and in vivo moisturising activity.
Materials and methods
Sorbitan oleate (HLB 4.3) and rice bran oil were kindly provided from Lipo do Brasil (Brazil). PEG-30 castor oil (HLB 11.7) was donated by Oxiteno (Brazil). The preservative DMDM Hydantoin (and) Iodopropynyl Butylcarbamate (Glydant® plus) was obtained from Chemyunion (Brazil), and the antioxidant Butyl Hydroxy Toluene (BHT) was purchased from Synth (Brazil).
Preparation of nanoemulsion
The following surfactant mixtures were evaluated to find a stable nanoemulsion formulation: polysorbate 80/sorbitan oleate, polysorbate 60/sorbitan oleate, ceteareth-20 OE/ceteth-2, PEG-15 castor oil/sorbitan oleate, PEG-30 castor oil/sorbitan oleate and PEG-40 castor oil/sorbitan oleate. The PEG-30 castor oil/sorbitan oleate (HLBresulting= 8.0) was the only surfactant combination that resulted in a stable formulation and therefore was chosen for further study.
The phase diagram method was used with different concentration of constituents to result in 42 different emulsion formulations. All emulsions were prepared according the Emulsion Phase Inversion (EPI) method, where the water and oil phases were heated separately at 75°C, the water phase was added into the oil phase (rice bran oil and surfactants) while stirring at 600 rpm (Fisatom, 713-Dmodel, Brazil), and the mixture was then cooled to 25°C while stirring.
Determination of nanoemulsion droplet size
The mean droplet size and polydispersity index of the nanoemulsions were determined by dynamic light scattering (DLS) (Zetasizer, modelo ZS, Malvern Instruments, UK). Measurements were performed at 25°C using a scattering angle of 90°. Samples were considered polydisperse when the polydispersity index was higher than 0.2 .
The preliminary stability of the nanoemulsion preparation was evaluated at 24 hours by centrifuge and thermal stress analyses. Stability was assessed by macroscopic emulsion observation and droplet size analysis. The purpose of these tests was to select a stable, low-surfactant formulation with a nanoemulsion-size droplet and stable physicochemical properties. The selected nanoemulsion was prepared in triplicate, and the samples were stored at 25 ± 2°C, 40 ± 2°C and 5 ± 2°C. Tests were performed at 24 hours, 7, 15, 30, 60 and 90 days after preparation. The analysis measurements were droplet size, pH value and electrical conductivity.
To perform the thermal stress test, nanoemulsions were heated in an ultra bath thermostat (Nova Técnica, Brazil) at temperatures ranging from 40 to 80°C. The temperature was increased by 5°C every 30 minutes. The nanoemulsions were centrifuged (Fanem 206-R, Brazil) at 1000, 2500 and 3500 rpm (70, 440 and 863 G, respectively) for 15 minutes in each rotation to accelerate possible instability phenomena. A pH meter (PM608 model - Analion, Brazil) was used to measure the pH of the nanoemulsions at 25 ± 2°C. The electrical conductivity was evaluated at 25 ± 2°C by a portable conductivity meter (mCA-150 model, Tecnopon, Brazil).
Irritant test in an organotypic model - HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane)
Classification of cumulative scores in the chorioallantoic membrane test (According Luepke 1985 )
In vivo assessment
Approval to conducted the in vivo studies was obtained from the Ethics Committee of Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil, under protocol number CEP/FCFRP n°. 147.
Seventeen Caucasian volunteers with healthy skin (all women, 20-29 years old), five patients with atopic dermatitis (all women, 21-30 years old), and four psoriasis patients (1 man and 3 women, 28-56 years old) without skin lesions on the forearm were included in the study. The forearm area was washed with mild soap two hours before the analysis. The volunteers were kept for 10 min in a controlled room at ambient temperature (25 ± 2°C; 29-34% relative humidity) before starting the assay. Baseline values were determined using a Corneometer CM 820, a Sebumeter SM 810 and a Skin-pH-meter PH 900 instruments (Courage &Khazaka, Köln, Germany) before applying the formulation. Then, 50 μL of nanoemulsion was applied to the right forearm in three rectangular areas of 13.80 cm2 and distributed for 20 seconds by rubbing the test area using a circular motion. Excess nanoemulsion was left on the skin. The treated skin of each patient was measured 30, 60, 90, 120 and 150 minutes after nanoemulsion application, with one measure for sebum content and triplicate measurements of electrical capacitance and skin pH.
Results and discussion
Preparation of nanoemulsion
Composition of formulation characterised as nanoemulsions
Rice bran oil (% w/w)
Sorbitan oleate/PEG-30 castor oil (% w/w)
Purified water (% w/w)
Droplet size (nm) ± (Standard Deviation)
91 ± 19
45 ± 12
69 ± 17
303 ± 32
121 ± 13
All of the nanoemulsions (Table 2) were stable when tested using the centrifugation test. The only formulation that showed signs of instability at high temperatures (70°C) was the nanoemulsion composed of 10:20:70 (oil, surfactants and water, respectively).
A surfactant concentration of 5.00% was not sufficient to form a nanoemulsion, even with smaller amounts of oil. The surfactant amount affects the stabilisation and size of the emulsion droplets. From the experimental results, the nanoemulsion that used the lowest possible surfactant concentration while still maintaining thermal stability, centrifugal stability and small droplet size was selected as the working formulation.
Based on these preliminary results, we chose a formulation composed of 10% rice bran oil, 10% surfactant blend and 80% water and proceeded with further stability tests and in vitro and in vivo evaluations.
The nanoemulsions had polydispersity index values below 0.2 throughout the 90-day testing period, indicating the high fidelity of the system (low polydispersity), which may reflect the overall stability of this formulation and synthesis method. Polydispersity values near 1.0 are indicative of a polydisperse system . The long term stability of nanoemulsions was previously evaluated and was also verified by stability studies conducted over three months. The W/O nanoemulsion produced by low energy emulsification showed no difference in droplet size over the study period at both 25°C and 4°C . The W/O nanoemulsion demonstrated high physical stability, corroborating our results for temperatures of 5 ± 2°C and 25 ± 2°C.
Low-energy emulsification is better at producing stable nanoemulsions than its higher energy counterpart. When nanoemulsions were prepared using a high pressure homogeniser, the droplet size was initially around 100 nm; however, the particles increased in size after 30 days at either 25 or 4°C. This phenomenon was attributed to the preparation method . The low-energy emulsification method used in our study showed high stability with respect to the droplet size and polydispersity index.
pH value determination
Monitoring the pH value is important for determining the emulsions' stability because pH changes indicate the occurrence of chemical reactions that can compromise the quality of the final product. Emulsions produced with vegetable oils may experience a decrease in pH due to the hydrolysis of fatty acid esters into free fatty acid degradation products .
Changes in the electrical conductivity can indicate nanoemulsion instability and may influence the nanoemulsion droplet size . In these studies, changes in electrical conductivity did not affect the nanoemulsion droplet size (Figure 3). It is difficult to assess the emulsion stability solely by electrical conductivity because the relationship between an increase in electrical conductivity and emulsion instability is not linear .
Thus, we could not conclusively determine the nanoemulsion's stability by this parameter. However, because the particle size and the pH value did not significantly change across different conditions, we considered our nanoemulsion to be stable. Nanoemulsion stability is a crucial parameter in determining the moisturising activity of the nanoemulsions in vivo.
Irritant test in an organotypic model - HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane)
Topical application products must have a low ocular/mucous membrane and a low dermal irritation potential. The irritation potential depends on the concentration of the substance as well as the chemical composition and the pH of the formulation .
Scores and assessments of irritation potential of nanoemulsions and surfactant solution tested in the chorioallantoic membrane test
Cumulative Score (average)
Previous studies showed that O/W microemulsions containing linoleic acid were only barely irritating in the HET-CAM test, indicated by a slight discoloration of the chorioallantoic membrane . The HET-CAM test showed that the nanoemulsion containing rice bran oil was essentially non-irritating.
In vivo assessment
The formulation composed of 10:10:80 (rice bran oil, surfactant blend and water) was chosen for the in vivo study due to its high stability and lack of irritation in the HET-CAM test. This nanoemulsion formulation was applied to volunteers with either normal or affected skin (atopic dermatitis or psoriasis).
The rice bran oil nanoemulsion significantly increased the skin hydration in volunteers suffering from atopic dermatitis and psoriasis. Although the skin hydration measurements should be conducted over a longer period of time, 8 - 24 h, this study indicates that the nanoemulsion may provide long-term skin hydration.
Skin pH determination
The nanoemulsion developed in this study using the phase diagram method was composed of 10% rice bran oil, 10% surfactants sorbitan oleate/PEG-30 castor oil, 0.05% antioxidants and 0.50% preservatives formulated in distilled water. The nanoemulsion was stable during the period of study and was found to be practically non-irritating in the organotypic HET-CAM model. When applied to the skin of volunteers, the nanoemulsion increased the relative hydration of the skin, the skin oiliness and maintained normal skin pH values. This nanoemulsion could serve as an alternative treatment for skin diseases such as atopic dermatitis and psoriasis.
This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) protocol numbers: 2009/01922-3, 2008/10382-0, 2009/05774-9, 2009/07817-7 and 2010/09618-9, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).
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