Preparation and Characterization of Resveratrol Nanoemulsions Stabilized by Self-assembly and Complex Coacervation Consisting of Sodium Alginate, Chitosan, and β-Cyclodextrin

Chitin was prepared by treating Chitinonecetes opilio with 15% hydrochloride solution at 50°C for 24 h (demineralization) and, subsequently, with 5% sodium hydroxide at 90°C for 12 h (deproteinization). Then chitin was treated with 60% sodium hydroxide at 140°C for 24 h to remove the acetyl residues (deacetylation). Next, the chitosan (degree of deacetylation 93%, molecular weight 330,000) was dissolved in a mixed aqueous solution of 20% hydrochloride solution and distilled water, after which the mixture was adjusted to pH 5.6 and stirred for 3 h. The reaction mixture was then subjected to a continuous membrane filtration process, after which permeate was freeze-dried.

To measure resveratrol solubility, various oil phases were selected (MCTs, limonene, corn oil, soybean oil, olive oil, and canola oil). The selection of oil phase was evaluated with analysis of particle size, degree of turbidity, and separation of oil phase through visual observation on the preparation of the nanoemulsion. Therefore, the optimum oil phase was selected with MCTs that could manufacture particle sizes of less than 100 nm and transparent in form. After ternary mixtures consisting of various types and compositions of oil phases were pre-tested.

Resveratrol (10 mg) was added to 1 mL of ethanol, followed by the addition of the oil phases at a 1:1 ratio, respectively. The mixture was stirred for 5 min and then subjected to vacuum evaporation at 50°C to remove the ethanol. The oil phases and Tweens^® 80 were then mixed together at a weight ratio of 1:3, followed by stirring for 5 min. To prepare the nanoemulsion, the mixture was accurately weighed (0.5 g) and diluted with deionized water (100 mL) as the aqueous phase, followed by magnetic stirring for 2 h and stabilization at 25°C for 24 h. Only with MCTs, measurement of the surfactant concentration was pre-tested using Tweens^® 80 at different ratios (resveratrol +MCTs:Tweens^® 80=1:1, 1:1.5, 1:2 and 1:3), based upon the results of our prior research involving the preparation of nanoemulsions using oleoresin capsicum (Choi et al., 2009). Tweens are widely used in food emulsion systems, primarily due to their relatively low toxicity, irritation potential and the ease with which they form micro- or nanoemulsions.

SLRNs consisted of oil droplets in an aqueous phase with the oil droplets being surrounded by a thin interfacial layer consisting of emulsifier molecules. SLRNs were prepared by self-assembly emulsification according to the following procedure. One of the six different oil phases used, MCTs produced highly dispersive emulsions consisting at all composition ratios of ethyl alcohol and resveratrol. Therefore, pre-mixture containing MCTs as the oil phase and Tweens^® 80 as surfactant was selected. Resveratrol (10 mg) was added to 1 mL of ethanol, followed by the addition of MCTs at a 1:1 (v/ w) ratio. The mixture was stirred for 5 min and then subjected to vacuum evaporation at 50°C to remove the ethanol. The oil phase and Tweens^® 80 were then mixed together at weight ratios of 1:1, 1:2, and 1:3 followed by stirring for 5 min (Table 1). To prepare the nanoemulsion, the mixtures were accurately weighed (0.5 g) and diluted with deionized water (100 mL). There is magnetically stirred for 2 h and stabilized at 25°C for 24 h. This primary emulsion was used for the preparation of SLRNs by dilution with deionized water.

Charged polyelectrolyte biopolymers are then added to the system of single layer nanoemulsions so that it absorbs to the droplet surface and produces DLRNs consisting of oil droplets coated by a biopolymer polyelectrolyte interface. DLRNs were prepared by complex coacervation with three different biopolymers: alginate (AN), chitosan (CN) and β-cyclodextrin (CYN). Alginate, chitosan and β-cyclodextrin solutions were prepared at concentrations of 0.05% in aqueous phase. Resveratrol (5 mg) was added to 1 mL of ethanol followed by the addition of MCTs at a 1:1 (v/w) ratio. The mixture was stirred for 5 min and then subjected to vacuum evaporation at 50°C to remove the ethanol. The oil phase and Tweens^® 80 were then mixed together at a weight ratio of 1:2, followed by stirring for 5 min. This mixture was accurately weighed (0.5 g), diluted in each of the above biopolymer solutions instead of deionized water, and then stirred for 2 h at 25°C. The nanoemulsions were then filtered with a 0.45 μm PVDF membrane filter (Whatman, Maidstone, Kent, UK) and stabilized at 25°C for 24 h before analysis.

The particle size distributions of the nanoemulsions were determined by photon correlation spectroscopy which analyzes the fluctuations in light scattering due to the Brownian motion of the particle using a Nanotrac NPA 250 (Microtrac Inc., Montgomeryville, PA, USA). Light scattering was monitored at 25°C at a 90° angle. The zeta-potentials of the nanoemulsions were measured by a ZetaPlus (Brookhaven Instruments Corp., Holtsville, NY, USA), which measures the distribution of the electrophoretric mobility of the particles using laser light scattering. 1.6 mL of each sample was poured into the electrophoretic cell. The particle size distribution and zetapotential value was analyzed at the optimum concentration range of Nanotrac and ZetaPlus through diluting between 10 and 100 times with deionized water. Each measurement was repeated 5 times on three different samples of the same composition.

The storage stability of SLRNs and DLRNs were evaluated by measuring the particle size and zeta-potential value during storage at 25°C for 4 weeks. The determination methods of particle size and zeta-potential value were same as above described method.

The stability of resveratrol from the SLRNs and DLRNs was determined by measuring the amount of resveratrol using liquid-liquid extraction. In this study, only CYN was used in the release test of resveratrol from DLRNs. The initial resveratrol content of the nanoemulsions was designated as 100%, whereas the decreasing resveratrol content was calculated via HLPC for 7 storage days at 25°C. The liquidliquid extraction of resveratrol was performed as follows. Nanoemulsions (1 mL) were mixed with 100% MeOH (2.5 mL) for 1 min, followed by the addition of hexane (10 mL) with stirring for 3 min. This mixture was maintained for 10 min at room temperature prior to the extraction of resveratrol. The methanol layer was recovered using a micro-syringe and then filtered with a 0.45 μm syringe filter. The extracted resveratrol was then analyzed by HPLC using a gradient controller unit 601-602 (FLOM, Tokyo, Japan) with an injection volume of 20 μL. Separation was performed on a C₁₈ column (Zorbax Eclipse XDB-C18, 4.6×250 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA). The mobile phase used was 100% acetonitrile, and the flow rate was 0.6 mL/min for 10 min. The UV/VIS detector S-3740 (Soma, Tokyo, Japan) was set at 308 nm. Standard curves were made using resveratrol dissolved in methanol (10, 30, 50 and 100 ppm).

All results are expressed as the mean±standard deviation. Statistical analyses of the data were carried out using SAS software (SAS Institute, Cary, NC, USA). Significant differences among groups of nanoemulsions were determined by Duncan’s multiple range test with p<0.05, p<0.01 and p<0.001, were considered statistically significant.

Resveratrol at a concentration of 5 mg/mL was solubilized in ethanol and then mixed with MCTs, limonene, corn oil, soybean oil, olive oil and canola oil at ratios of 1:1. These resulting mixtures were then added to Tweens^® 80 at ratios of 1:3 (w/w). The emulsion was prepared by dissolving the mixtures with water at concentrations of 0.5%. When the ethanol-resveratrol solution was mixed with corn oil, soybean oil, olive oil, and canola oil, it became cloudy and separated from the oil phase. Additionally, the particle size was found to be within 191-936 nm (Table 2). In contrast, MCTs and limonene showed particle sizes of about 16.15 and 12.11 nm, respec- tively, which implies they can serve as the oil phase for the production of resveratrol-containing nanoemulsions. Resveratrol does not completely dissolve in buffering solutions with pHs of 4-5, but it does show relatively high solubility in coconut oil as well as glycerol (Hung et al., 2006). Additionally, the use of limonene is limited due to its characteristic flavor. Therefore, MCTs are considered to be the appropriate oil phase for the production of nanoemulsions containing resveratrol. In addition, due to its high solubility in ethanol, resveratrol could be used for the manufacture of nanoemulsions containing high concentrations of resveratrol.

Table 1 displays the particle sizes and resveratrol contents of nanoemulsions based on various ratios of resveratrol and MCTs with Tweens^® 80. The highest resveratrol content (250 μg/mL) was produced when the resveratrol and MCTs mixture was mixed with Tweens^® 80 at a 1:3 ratio (Resveratrol+MCTs : Tweens^® 80 : water composition ratio of 5 : 15 : 80, w/w) (Table 1). In addition, when resveratrol and MCTs were mixed with Tweens^® 80 at a 1:2 ratio, the particle size was 30 nm and the resveratrol content was 83.3 μg/mL (Resveratrol+MCTs : Tweens^® 80 : water composition ratio of 1.67 : 3.33 : 95, w/w). The particle sizes of the nanoemulsions after mixing of resveratrol and MCTs with Tweens^® 80 at ratios of 1:1, 1:1.5, 1:2 and 1:3 are presented in Table 3. When the mixing ratio was 1:2 or 1:3, the particle sizes of the nanoemulsions were 16.3 and 16.2 nm, respectively. However, mixing ratios of 1:1 and 1:1.5 resulted in sizes of 231 and 554 nm, respectively, which indicates that the emulsions must be unstable if subjected to storage. Based on the results, the appropriate mixing ratio of Tweens^® 80 for the production of resveratrolcontaining nanoemulsions is considered to be two times that of the weight of the resveratrol and MCTs mixture. A phase diagram of the three-component resveratrol+MCTs/Tweens^® 80/water system at ratios of 1:1, 1:1.5, 1:2 and 1:3 is presented in Fig. 1. This composition ratio of Tweens^® 80 could be used as a quality indicator related to the allowance limit of additives in the food industry. The phase behavior of such samples was determined only after sharp interfaces become visible. When the mixing ratio of the resveratrol and MCTs mixture with Tweens^® 80 was 1:2 or 1:3 in 60% water, nanoemulsions formed with particle sizes of less than 100 nm, which is indicated as the white region in the diagram. There were exhibited white areas as one-phase nanoemulsions region (below 100 nm) and gray areas as multiple-phase nanoemulsions (between 100 and 700 nm), respectively. Because nano-sized emulsions below 100 nm absorb light, these samples will have a transparent appearance. This differs from the micro-sized emulsions larger than 300 nm which will scatter light sufficiently to allow them to be seen by naked eye and have a cloudy white appearance.

Fig. 1. Phase diagram of nanoemulsions formed by self-assembly of the water/resveratrol+MCTs/Tweens^® 80 system. Res+ MCTs:T: resveratrol+medium-chain riaylglycerols:Tweens^® 80. White area is the region of the nanoemulsions ranging below 100 nm.

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To confirm the stability of SLRNs, the mixing ratios of the resveratrol and MCTs mixture with Tweens^® 80 were varied as 1:1, 1:2 and 1:3, and the particle sizes of the resveratrol nanoemulsions during 4 weeks of storage at 25°C were measured (Table 4). When the mixing ratio was 1:1, the particle sizes of emulsions were 136-176 nm at mixture concentrations of 0.5, 1 and 3% (w/w); whereas, the particle sizes of 251-655 nm were observed at concentrations of 5, 10, and 20% (w/w) at 0 day. When the mixing ratio was 1:2, particle sizes of 27, 25, 24, and 30 nm were observed to be concentrations of 0.5, 1, 3, and 5%, respectively, showing that particle size was reduced by increasing the amount of Tweens^® 80 and maintaining a stable state during storage. A mixing ratio of 1:3 resulted in particle size of less than 15 nm when the Tweens^® 80 was added at concentrations of 0.5, 1, 3, 5, and 10% (w/w); these nanoemulsions were stable throughout storage. For the production of nanoemulsions, the amount of Tweens^® 80 added has large effects on particle size reduction and stability of the nanoemulsion (Choi et al., 2009). The storage stabilities of the resveratrol nanoemulsions prepared by mixing resveratrol and MCTs with Tweens^® 80 with a ratio of 1:2 are presented in Table 5. The DLRNs prepared with alginate and chitosan were unstable and showed slight increases in particle size during the 4 week storage period. However, the DLRNs prepared with β-cyclodextrin were more stable with particle sizes of less than 40 nm during storage. For the SLRNs, they were relatively stable during storage, but their stability was slightly reduced after 2 weeks of storage. These differences in stability between the SLRNs and DLRNs may be due to their initial particle sizes governed by their composition that included surfactants, bio-polymers (e.g. alginate and chitosan), and lipids.

The droplets in most nanoemulsions have an appreciable electrical charge; therefore, electrostatic interactions may play an important role in determining their overall stability and physicochemical properties. The zeta potential, which represents the electric charge between the shear plane of the final external layer and the bulk solution, has been used to estimate the property of dispersions. The zeta potentials of the resveratrol nanoemulsions prepared by mixing resveratrol and MCTs with Tweens^® 80 at a ratio of 1:2 are indicated in Table 6. The zeta potential of the DLRN prepared with chitosan was 0-37.7 mV during storage due to the positive charge of the chitosan molecules coated on the surface of nanoemulsions. In contrast, the zeta potential of the DLRN prepared with alginate was -14.4 - -24.8 mV due to the negative charge of the alginate molecules. Meanwhile, the zeta potential of the DLRN prepared with β-cyclodextrin resulted in a stable negative value ranging from -5.6 - -14.6 mV. The droplets of most nanoemulsions have an appreciable electrical charge, suggesting electrostatic interactions may play an important role in determining overall stability and physicochemical properties (Nielloud & Marti-Mestres, 2000). The zeta potential represented by the electric charge between the shear plane of the final external layer and the bulk solution, is used to estimate the extent of dispersion. Furthermore, this factor is influenced by the electrical characteristics between nanoemulsion particles.

The stability of resveratrol from the DLRN prepared with β- cyclodextrin is shown in Table 7. The resveratrol content of the DLRN prepared with β-cyclodextrin was much higher than the SLRN, which it was reduced to 94.6% after 7 days. However, for the SLRN, the content of resveratrol was reduced to about 88.5% in the same storage period, demonstrating lower stability compared to the DLRN. This result indicates that delayed resveratrol release when resveratrol was present in an emulsion or aqueous micelle system. Moreover, the stability of resveratrol is related with its reduction in the order of aqueous micelle system > tocol emulsion > lipid emulsion (Hung et al., 2007). Therefore, the DLRNs prepared with β- cyclodextrin have potential as a nutraceutical delivery system for the purpose of increasing the stability and utility of physiological active compositions obtained from functional foods within the body. Controlled release of resveratrol is dependent on pH with slower release kinetics at higher pH values due to the protonated amino groups of chitosan resulting in a soluble, positively-charged polysaccharide that causes fast swelling in acidic medium (Peng et al., 2010).

The biopolymers such as chitosan and alginate were increased the stability of resveratrol via complexation with DLRNs. However, the DLRNs prepared with β-cyclodextrin resulted in the highest stability. Therefore, β-cyclodextrin was the best in maintaining the stability of the resveratrol nanoemulsions. In addition, β-cyclodextrin is available in solid and powder form, making it ideal in a nutraceutical delivery system.