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Development of a topical applied functional food formulation: Adlay bran oil nanoemulgel
LWT - Food Science and Technology 117 (2020) 108619
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Development of a topical applied functional food formulation: Adlay bran oil nanoemulgel
T
Wen-Chang Changa,b,1, Yin-Ting Hua,1, Qingrong Huangc, Shu-Chen Hsieha,∗∗, Yuwen Tinga,∗ a
Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City, Taiwan Department of Food Science, National Chiayi University, No. 300, Syuefu Rd., Chiayi City, Taiwan c Food Science Department, Rutgers University, 65 Dudley Rd., New Brunswick, NJ, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adlay bran oil Functional food By product Topical delivery Emulgel
Using bioactive ingredient from natural food sources to treat chronic health problems has become an emerging trend. Compare to oral consumption, topical application is still the preferred route for administrating these active food ingredients to skin for direct treatment of dermal diseases. Emulgel, a polymeric emulsion system, possesses the properties of both emulsions and hydrogel. Thus, a polymeric emulsion dosage could encapsulate both hydrophilic and hydrophobic compounds and improves their storage stability and cellular uptake. Extracted from the by-product of adlay processing, adlay bran oil is found to ameliorate many skin related diseases. Through the construction of a pseudo-ternary phase diagram, a formulation that contains 60% water, 28% adlay bran oil, and 12% Tween 80 was selected since it generates nano-droplets while still maintaining a sufficient surface energy for better stability. Two cycles of 1000 bar pressure treatment were required to produce nanoemulsion using high-pressure homogenizer. The addition of Pluronic® F-127 successfully transformed nanoemulsion into nanoemulgel. Depending on the gelator concentration, the sol-gel transition temperature varies from 20 to 30 °C. Apart from nanoemulsion facilitating higher permeation rate, nanoemulgel promoted the accumulation of bioactive component on the skin is a more suitable functional food formulation for topical uses.
1. Introduction Adlay bran oil (ABO), obtained from pressing the bran of adlay, or Job's tears (Coix lachrymal-jobi L. var.ma-yuen Stapf), is the main byproduct of the adlay refining process. It has good antioxidant, anti-inflammatory, anti-pigmentation, and anti-cancer effects due to its high content of bioactive components (C.-J. Huang et al., 2015). The phytochemical constituents isolated from adlay bran can be divided into nine main categories—namely, phenolic acids, phenolic esters, phenolic aldehydes, flavonoids, triterpenoids, sterols, lactams, lactones, and lignans. (C.-J. Huang et al., 2015). Using ABO to develop health-promoting formulations is desirable because it reduces production waste and increases the economic value of adlay processing. As the largest organ of the human body, the skin is the most critical barrier involved in preventing the invasion of pathogens as well regulating body temperature. However, the skin is vulnerable to skin cancer, one of the most common cancers worldwide, as well as other diseases. The use of emulsions can enhance the efficacy of bioactive
ingredients used to treat the skin diseases. According to previous research, an emulsion is better absorbed when it is processed to a smaller size and is allowed to stay in contact with the skin longer (Pouton, 1985). Therefore, it is desirable to reduce the size of the emulsion droplets and to increase surface retention time when developing emulsion products for skin use. The formation of an emulsion requires the intervention of an emulsifier to facilitate the uniform dispersion of two immiscible liquids. Selecting a suitable emulsifier can promote the formation of a homogeneous system with better storage stability (Alexander et al., 2013). The stability of an emulsion can be extended by processing it into a gelled system by incorporating a gelling agent (Mohamed, 2004). In this sense, oil-in-water (O/W) or water-in-oil (W/O) emulsions form a macromolecular network structure that prevents the aggregation of the dispersed phase and, thus, improves the stability of the continuous phase. An emulgel is an emulsion mixed with a gelling agent and it is a favorable delivery system for bioactive ingredients when a longer retention time is required (Devada, Jain, Vyas, & Jain, 2011).
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (S.-C. Hsieh), [email protected] (Y. Ting). 1 These authors contribute equally. ∗∗
https://doi.org/10.1016/j.lwt.2019.108619 Received 14 February 2019; Received in revised form 9 September 2019; Accepted 10 September 2019 Available online 11 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 117 (2020) 108619
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Topical administration using delivery systems such as emulsions, ointments, and hydrogels is recommended for many skin problems. However, these topical dosages all have one or more drawbacks, including poor loading concentration, low diffusion coefficient, insufficient stability, and their limitation to the delivery of only hydrophiles or lipophiles. To enhance the functionality of an available topical delivery system, a two-step process that transforms the liquid emulsion into a semisolid emulsion gel has been developed. Emulgel, a polymeric emulsion system, has the advantages of both an emulsion and a hydrogel system. With the proper design, an emulgel system with a shear thinning property could reproduce not only the excellent stability of a hydrogel, but also the superior skin permeability of liquid emulsions (Devada et al., 2011). The wide variety of gelling agents that are soluble in either oil or aqueous environments allow the encapsulation of both hydrophobic and hydrophilic components in the O/W and W/O emulsions, respectively. Moreover, an emulgel that has a higher viscosity than the liquid emulsion system can increase the retention time of active components on the skin surface and promote better absorption (Mohamed, 2004). Overall, emulgels are a favorable formulation in the percutaneous absorption of both hydrophilic and lipophilic components because they offer alternative phases (dispersed and a continuous), higher skin permeability, better application and storage stability, and the ability to control the release rate. As bioactives isolated from adlay are effective against many skinrelated problems, it is rational and economical to use the oil extracted from adlay bran for skin product development. Specifically, adlay bioactives have been documented to be effective in inhibiting cellular tyrosinase activity and melanin production and, thus, preventing hyperpigmentation, which results from hormonal changes, aging, skin diseases, or injuries (H.-C. Huang, Hsieh, Niu, & Chang, 2014). In this study, an emulsion-based delivery system, including a conventional emulsion (CE) (d > 1 μm), nanoemulsion (NE) (d < 500 nm) (Chime, Kenechukwu, & Attama, 2014; Qian, Decker, Xiao, & McClements, 2012; Zhou, Wang, Cheung, Guo, & Jia, 2008), and nanoemulgel (NG), was prepared and optimized for skin use. The gelling agent selected for the preparation of nanoemulgel system was Pluronic F-127 (PF-127), which is one of the poloxamer ABA block polymer. The special chemical structure of PF-127 makes it more readily soluble in cold than in hot water and, thus, would allow it to maintain a gel-like property at body temperature. The physicochemical properties and percutaneous absorption rate of the prepared skin formulations were then studied systematically. This study is the first to report the production of an emulsion system using ABO. Our results not only demonstrate the potential to re-use a manufacturing by-product but also serve as an excellent reference for selecting a different delivery system for topical applications.
ABO was simultaneously filtered, collected using a serum bottle and stored in the dark at 4 °C until future application. The oil extraction yield was approximately 20%.
2. Materials and methods
To avoid multiple scattering effects, samples were diluted 500-fold with DI water before measurement. After dilution, the samples were analyzed by the dynamic light scattering method (Nanotrac 150, Microtrac, Montgomeryville, PA, USA). The droplet size (nm) was estimated as the mean diameter (in microns) of the volume distribution from 3 replicates:
2.3. Construction of pseudo-ternary phase diagram The pseudo-ternary phase diagram of the ABO, Tween 80®, and deionized water was constructed using a method reported by Xi et al. with minor modifications (Xi et al., 2009). Briefly, the oil phase was prepared by mixing ABO and Tween 80 at weight ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. The prepared oil phase was then combined with deionized (DI) water by the aqueous titration method, in which 100 μL of DI water was gradually added each time and the proportion of DI water was progressively increased. After homogenizing for 2 min with a high-speed homogenizer (T25 digital, IKA, Germany) at 25,000 rpm, samples were left standing in the dark for 1 h at room temperature (~25 °C). After 1 h, the physical status of each emulsion sample was observed and recorded.
2.4. Preparation of emulsion and nanoemulsion (NE) samples To prepare the emulsion system, ABO and Tween 80 were added to the glass bottle at a weight ratio of 9:1, 8:2, 7:3, and 6:4, mixed well and, combined with DI water. The conventional emulsion (CE) was prepared by homogenizing the mixture for 2 min with a high-speed homogenizer (T25 digital, IKA, Germany) at 25,000 rpm. To produce a nanoemulsion, the emulsion was subjected to a high-pressure homogenizer (N-2 Nanolyzer, Gogene Co., Taiwan) at a pressure of 1000 bar. To facilitate observation, ABO was first combined with 1% (v/v) Oil red O stain before preparing the CE and NE samples used for the construction of pseudo-ternary phase diagram and the study of accelerated storage stability.
2.5. Preparation of nanoemulgels (NGs) Based on the result obtained from the pseudo-ternary diagram, the optimum NE with the smallest droplet size was selected for further preparation of the NG. The gelling agent (Pluronic® F-127) was first dissolved in DI water and then mixed with NE at final concentration of 10%, 12%, 15%, 18%, 20%, and 22% in a glass bottle. The gel structure appeared after being stirred on a magnatic stirr plate at 4 °C for 24 h.
2.6. Droplets size analysis
2.1. Chemical reagents Folin-Ciocalteu phenol reagent and sodium carbonate were purchased from Merck (Darmstadt, Germany). Pluronic® F-127 (PF-127), Tween® 80 and Oil red O were obtained from Sigma-Aldrich (St. Louis, MO, USA). Kojic acid was purchased from TCI (Tokyo, Japan). All other chemicals and solvents were of analytical grade. Deionized water was prepared with a Milli-Q water purification system (Millipore, USA).
MV = ΣVidi / ΣVi
(1)
where MV is the mean volume diameter (μm), V is the volume percent (%) of measured droplet size and d is the droplet diameter (μm). All results are present as the average measurement of samples produced from 3 separate batches.
2.2. Preparation of adlay bran oil (ABO) Adlay (Taichung selective No. 4) cultivated and taxonomically identified by the Taichung District Agricultural Improvement Station (Taichung, Taiwan) was selected for the experiment. The oil was obtained by pressing only the adlay bran (the outer shell) with a lowtemperature presser (SP-230,SHANQ JER industries CO. Ltd., Taiwan). The adlay bran was packed in a filter cloth and pressed in two stages.
2.7. Zeta potential analysis Each sample was diluted 500-fold with DI water before being subjected to zeta potential analysis (Zetasizer Nano Z, Malvern Instruments Co., Ltd., Worcestershire, United Kingdom). 2
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collected from the receiving chamber of the Franz diffusion cell was analyzed by a microplate reader (Epoch™ Microplate Spectrophotometer, BioTek Instruments, Winooski, Vermont, USA) as reported by Singleton et al. (Singleton & Rossi, 1965). The standard curve was constructed using 0, 0.625, 1.25, 2.5, 5, 10, 25, and 50 μg/ mL of gallic acid in a volume of a 25 μL per well in a 96-well plate. Later, 25 μL of Folin-Ciocalteu reagent was added to each well and allowed to react in the dark for 5 min, then 50 μL of DI water and 150 μL of 5% Na2CO3 solution were added to each well and again incubated for 1 h in dark. The reader then determined the light absorption value at 750 nm. The total phenolic content in the sample was determined from the standard curve and expressed in terms of gallic acid equivalent concentration (μg/mL).
2.8. Rheological analysis A rheometer (AR 2000ex, TA Instruments, New Castle, DE, USA) was used to measure the rheological behavior of the prepared samples. Sample viscosity was measured using a 40 mm diameter parallel plate set at a 2 mm gap distance at 25 °C. Data were recorded every 5 s for a total of 2 min, during which angular frequency ω range from 1 to 200 rad/s was gradually applied to the sample. In the stress sweep test, changes on the storage modulus (G′) were studied when 0.1–1000 Pa of the oscillation stress was applied to samples at a fixed frequency of 1 Hz. Finally, the gelling behavior of NG over a temperature ramp was also determined through a temperature increase from 4 to 80 °C at a frequency of 1 Hz and 0.5% strain. Values of storage modulus (G′) and loss modulus (G″) were obtained every 30 s and Tan δ was calculated accordingly. The rate of temperature increase and the rate of temperature drop were both 1 °C per minute.
2.12. Statistical analysis All tests were conducted in at least 3 replicates and were presented as the average of measurements. The data were analyzed using a oneway analysis of variance and Duncan's new multiple-range test using SAS statistical software package (SAS Institute, Cary, NC, USA). P values of < 0.05 were considered significant. The data are shown as the mean ± standard deviation (SD).
2.9. Accelerated storage test Storage stability is an important quality parameter commonly studied by investigators when developing new formulations. Instead of the real-time stability test, the accelerated test using elevated stress conditions is more commonly applied to rapidly predict the degradation rate of candidate formulations. The test we used was carried out with a slight modification based on the method of Restu et al. and Smrity et al. (Restu, Sampora, Meliana, & Haryono, 2015; Smrity, Saifuddin, & Sultana, 2016). To complete one accelerated storage cycle, an equal amount of CE, NE, and NG were put into separate glass bottles, capped, refrigerated at 4 °C for 24 h, and then moved to a 45 °C chamber to store for another day. The sample used for the accelerated storage test were observed for any sign of physical changes. In addition, the droplet size and zeta potential of all samples were determined at the end of each of five temperature cycles. The sample was considered stable if it remained physically unchanged and maintained constant droplet size and surface charges.
3. Results and discussion 3.1. Construction of pseudo-ternary phase diagram By constructing a pseudo-ternary phase diagram, all possible mixing ratios of the emulsifier and the organic and aqueous phases were studied systematically, resulting in the elucidation of formulations of various physicochemical properties. Using a titration method, we investigated the existence of a stable emulsion region where the organic phase was composed of 10–90% of the emulsifier (Tween 80) in ABO and was titrated by the aqueous phase (DI water) at various ratios (Fig. 1A). Starting at 9-to-1 ratios of organic-to-aqueous phases, stable water-in-oil (W/O) emulsions were formed when the organic phase contained more than 60% of Tween 80. As more of the water was added, the emulsion systems underwent a liquid-to-semisolid transition, during which gel-like structures were formed (indicated by the solid black circle in the diagram). Later, the semi-solid formulations experienced another transition back to the liquid form, when the percentage of the aqueous phase was increased within the range of 35–60% (w/w) in the systems containing lower to a higher proportion of emulsifier, respectively. The result is consistent with previously published results, describing a linear correlation between concentration and emulsification power, which was indicated by more stable emulsion formulations that were formed with higher contents of Tween 80 (Chen & Tao, 2005; Fernandez, André, Rieger, & Kühnle, 2004). To produce droplets in the nanoscopic range, emulsion systems should have the suitable flow property enabling them to pass through the high energy delivering instrument, such as a high-pressure homogenizer. The flow property (commonly known as viscosity) of an emulsion system is inversely correlated to the oil content of the formulation (Floury, Desrumaux, & Lardieres, 2000). However, in the case of ABO as the organic phase, it is desirable to have a higher oil content, because of all the bioactive components contained within. Therefore, it is desirable to maximize the oil content in the emulsion while still allowing its smooth passage through the high-energy homogenizer. The semi-solid formulations, although containing a higher amount of oil, have poor fluidity and, thus, were not selected for further processing into a nanoemulsion. According to the phase diagram, all formulations containing more than 60% water had liquid-like fluidity regardless of the emulsifier content in its organic phase. Tween 80 is simultaneously an emulsifying and gelling agent, thus its concentration significantly influences the viscosity of a formulation (Prieto & Calvo, 2013; Sagiri, Behera, Sudheep, & Pal, 2012). In Fig. 1B,
2.10. Percutaneous absorption test The skin absorption rate was determined in vitro using a Franz diffusion cell. Briefly, the effective absorption area of the device was first covered by a 6.25 cm2 piece of back pigskin (0.3–0.5 mm thick) and then assembled with an upper and a lower chamber using a metal clamp. The skin from a 3-4-month-old pathogen-free miniature pig was directly purchased from the Agricultural Technology Research Institute (Taipei, Taiwan). Approximately 0.5 g of the formulation prepared for skin absorption was placed in the upper chamber. At 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h, 500 μL of sample was collected from the lower receiving chamber, which was initially filled with 11 mL of PBS. The concentrations of phenolic compounds from the collected samples were analyzed using a spectrophotometric method that will be described in a later section. Compound penetration volume per unit time was calculated using the following formula: Wn (μg) = Cn × V + Vs × (C1 + C2 + C3 + … …..Cn-1)
(2)
where Wn is the weight of the total phenolic compounds in the receptor fluid during the nth sampling, Cn is the concentration of the total polyphenolic compounds in the receptor fluid during the nth sampling, V is the total volume of the receptor fluid, and Vs is the volume of the receptor fluid during each sampling. Finally, the cumulative compound penetration volumes were plotted against time to study the absorption kinetics. All experiments were repeated at least three times (n = 3). 2.11. Total phenolic contents The concentration of the phenolic compound in the samples 3
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Fig. 1. (A) Pseudo-ternary phase diagram and (B) viscosity of the emulsion composed of adlay bran oil, Tween 80, and water (60%). The results are expressed as the mean ± SD of each group (n = 3).
Despite the fact that a nonionic emulsifier was used, the surface of the emulsion droplet was found to be negatively charged due to the spontaneous charging of the oil-water interface, to which hydroxyl ions released by the dissociated water molecules were absorbed (Marinova et al., 1996). This phenomenon is supported by the negative zeta potential values that were detected on the surface of all formations (Table 1). However, the concentration of emulsifier did not significantly affect the magnitude of the absolute surface charge until the droplet size was further reduced to less than 300 nm by a high-pressure homogenizer. The resulting increase in surface area covered with uncharged emulsifier led to a reduction in the number of absorption sites for the hydroxyl molecule, thus lowering the magnitude of the surface charges. That is, high-pressure homogenization not only effectively reduced the droplet size but also significantly decreased the amount of surface charges on the droplets as indicated by the zeta potential measurement. All formulations except for the one that contained 40% Tween 80 in the organic phase maintained its zeta-potential value of more than (−) 30 mV. This observation could be attributed to the excessive surfactant absorption on the droplet surface forming a thick protection layer that prevented the hydrogen bonding between the absorbed hydroxyl group and a water molecule. As stated previously, if the absolute magnitude of the zeta potential is higher than 30 mV, then it can contribute to the stability of the colloidal system (Patel & Agrawal, 2011). Thus, to enhance the systemic stability, it is desirable to select a formulation that can produce an emulsion of nanoscopicsized droplets and with a more substantial surface charge.
a positive relationship between the proportion of Tween 80 in the organic phase and viscosity of the formulation was shown when water content was kept constant at 60%. Since the formulations that could successfully pass through the available high-pressure homogenizer were only those with a viscosity of less than 1 Pa s, the oil phase of the emulsion system could not contain more than 40% of the Tween 80. 3.2. Properties of the emulsion (CE) and nanoemulsion (NE) To find the optimum mixing ratio, the physical properties of formulations containing 60% aqueous phase and 40% organic phase were analyzed. Based on the rheological analysis, formulations with an organic phase containing 10%–40% of Tween 80 in the ABO could be further processed using a high-pressure homogenizer. As indicated by Stoke's law, the rate at which an emulsion system becomes unstable is positively correlated with its droplet size. After subjecting the system to high-speed homogenization, the droplet size of formulations containing 10%, 20%, 30%, and 40% of Tween 80 in the oil phase were approximately 1345.50, 951.00, 610.50, 590.00 nm, respectively. This result agrees with a previous investigation where the ratio of grease to Tween 80 had a significant effect on the droplet diameter of the emulsion system (Chen & Tao, 2005). After subjecting the system to high-pressure homogenization, the droplet size of all formulations decreased by 4-12-fold, depending on the concentration of the emulsifier. However, only formulations containing 30% and 40% of Tween 80 in the organic phase contained droplets with an average size of less than 100 nm (Table 1), which is a strict requirement for the nanoscopic product (Bhattacharjee, 2016).
3.3. Effect of homogenization parameters on the droplet diameter Table 1 Physical properties of adlay oil emulsion and nanoemulsion with different adlay oil and Tween 80 ratios. Properties D (nm)
ζ (mV)
Percentage of Tween 80 (%) 10 20 30 40 10 20 30 40
Emulsion 1345.5 ± 246.9 951.0 ± 27.3b 610.5 ± 68.8c 590.0 ± 110.0c −48.4 ± 0.8a −44.2 ± 1.1b −46.3 ± 1.7ab −40.3 ± 0.8c
The size of the emulsion droplet is affected by the processing parameters during high-pressure homogenization. Among all influencing factors, the level of applied pressure and number of input cycles most significantly affect droplet diameter (Qian & McClements, 2011). Based on previous section results, a formulation containing 60% DI water, 28% ABO, and 12% Tween 80 was chosen to manufacture NE. The selected formulation was subjected to processing pressures of 500, 1000, and 1500 bars for 1–5 cycles. After the first cycle, the emulsion droplet size was significantly reduced under all pressure magnitudes. Comparing the three processing conditions, an applied pressure of 500 bar was less efficient since it produced larger droplets than applied pressures of 1000 and 1500 bars at all processing cycles (Table 2). Although the 1500 bar pressure could produce the smallest emulsion droplets within the 1st cycle, it also generated excessive heat that caused rapid water evaporation and, thus, increased the chance of
Nanoemulsion a
214.4 ± 14.0a 197.1 ± 14.1b 73.7 ± 9.0c 46.0 ± 2.3d −34.2 ± 1.2a −36.3 ± 1.2a −33.0 ± 2.8a −25.8 ± 0.9b
The results are expressed as the mean ± SD of each group (n = 3). Values with different letters (a-d) between columns indicate a statistically significant difference among emulsion and nanoemulsion (p < 0.05). D is particle size, ζ is zeta-potential, and μ is viscosity at 100 s−1 of shear rate. 4
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Table 2 Effect of processing pressure and cycles on the droplet diameter. Droplet diameter (nm) Pressure (bar) 500 1000 1500
Cycle 1 127.8 ± 8.0a 78.1 ± 3.4a 66.9 ± 6.8a
Cycle 2 94.8 ± 5.1b 61.6 ± 4.2b 59.7 ± 5.2a
Cycle 3 83.0 ± 4.3cd 61.1 ± 1.4b 61.2 ± 3.4a
Cycle 4 90.3 ± 3.3bc 60.5 ± 2.7b 61.0 ± 4.8a
Cycle 5 77.2 ± 3.8d 63.8 ± 2.3b 66.5 ± 1.8a
The results are expressed as the mean ± SD of each group (n = 4). Values with different letters (a-d) indicate a statistically significant difference (p < 0.05) between the number of cycles under 500, 1000, or 1500 bar pressures.
capacity than other carrier systems (Mou et al., 2008). In the specific case of topical application, sufficient formulation viscosity is necessary for sufficient skin contact time, during which the bioactive compounds contained within can be released and permeate into the skin (Bhattacharjee, 2016). Thus, a suitable polymeric gelling agent is usually added to the liquid formulation to enhance its stability and skin retention time by modifying its rheological property (Valenta & Schultz, 2004). Pluronic F-127 (PF-127), a non-ionic gelling agent, forms monomolecular micelles and multimolecular aggregates (micelles ordered into a lattice) at low and high concentrations, respectively. Thus, a higher concentration (> 15%) of PF-127 is required for the construction of the gel structure (Patel & Agrawal, 2011). In this investigation, the emulsion system was only starting to gel when the concentration of PF-127 was higher than 18% (w/w). A similar phenomenon was reported by Nie et al., who showed that 18% of PF-127 is required for the preparation of a liposomal gel (Hsu & Nacu, 2003). Besides concentration, the gelation property of PF-127 is also affected by temperature. An aqueous solution containing 20–30% (w/w) of PF-127 shows an interesting reversible thermal gelation behavior (Lenaerts, Triqueneaux, Quartern, Rieg-Falson, & Couvreur, 1987; Miyazaki, Takeuchi, Yokouchi, & Takada, 1984), i.e., the solution is liquid at a refrigerated temperature and will slowly gel as the temperature is increased. The temperature at which the PF-127-containing formulations change from the liquid state to a gel state is significantly influenced by the concentration. The sol-gel transition occurred when the temperature was increased to 30.6 ± 0.3, 25.5 ± 0.6, and 20.5 ± 0.1 °C for formulations containing 18%, 20% or 22% of PF-127, respectively (Fig. 3A). The experimental data supports the inverse relationship between the PF-127 concentration and the sol-gel transition temperature. Thus, thermally reversible properties were also observed, as would be expected from the results of a previous investigation (Liu, Sun, Li, Liu, & Xu, 2006). The formation of a polymer micelle is mainly driven by the hydrophobic interaction between the polyoxyethylene-polyoxypropylene copolymers in PF-127 (Hunter, 2001). Thus, any factor that interferes with the hydrophobic interaction between PF-127 molecules will significantly decrease micelle formation and, thus, disturb the molecular arrangement into an ordered lattice structure (IWH, 1998.). Comparing PF-127 concentrations, the inclusion of NE in the system significantly raised the sol-gel transition temperature when the PF-127 concentration was at 18% and 20% (Fig. 3B). This phenomenon implies that PF127 can be absorbed onto the droplet surface and, thus, its efficient micelle aggregation is disturbed by the presence of an emulsion. When the temperature was further increased, more of the hydrophilic chains of copolymer became desolvated, which favors the hydrophobic interaction (Miller & Drabik, 1984). Thus, the addition of NE was less influential to the sol-gel transition temperature of PF-127 when its concentration was 22%. Spreadability, a fundamental rheological property, was studied by monitoring the change in the storage modulus (G′) when applying a sweep of shear stress to a topical dosage. As shown in Fig. 3C, the G′ value and the yield stress of NG went up as the PF-127 concentration increased. That is, the rigidity and strength of NG increased as the concentration of PF-127 increased. The result indicates that the formulation that contains more PF-127 can improve its skin retention
machine blockage due to higher sample viscosity. Under 1000-bar pressure, more than two runs through the homogenizer was necessary since the sample droplet size did not change significantly after the 2nd run (Table 2). Given the alternatives, we therefore selected an applied pressure of 1000 bars as the final processing pressure for high-pressure homogenization, even though it requires two processing cycles to generate satisfactory nanodroplets,. 3.4. Effect of high-pressure processing on storage stability To study the system stability, emulsion samples before and after high-pressure processing were studied using the accelerated storage test. Before high-pressure treatment, the CE (diameter > 500 nm) phase separated after being subjected to the accelerated test for one day, whereas the NE (diameter < 100 nm) maintained its stable 1phase structure even after ten days of storage under extreme conditions. During the experiment, there was no significant change in the droplet size of NE, further indicating good storage stability (Fig. 2). Results of the accelerated storage test show that high-pressure processing effectively reduces the droplet diameter of the emulsion, while at the same time enhancing the stability of the system. This result is consistent with Stoke's law, the droplet diameter is inversely related to its velocity of becoming unstable. More evidance on the enhance stability thorugh decreasing the droplet size was also found in previously published literatures (Abismaıl, Canselier, Wilhelm, Delmas, & Gourdon, 1999; Vladisavljević & Schubert, 2003). 3.5. Physical properties of adlay oil nanoemulgel Unlike microemulsions, an NE is a non-equilibrium system and will eventually become unstable after storage for an extended period of time. However, NE is commonly used in pharmaceutical and cosmetic products because it offers several significant advantages, such as lower skin irritation, better membrane permeability, and higher loading
Fig. 2. Particle size of adlay bran oil nanoemulsion measured during the accelerated storage test. 5
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Fig. 3. Storage modulus (G′) and loss modulus (G″) of a nanoemulsion gel containing (A-a) 18%, (A-b) 20%, and (A-c) 22% of PF-127 as a function of temperature. (B) Gelation temperature of the nanoemulsion gel contained various amount of PF-127. (C) Storage modulus (G′) of nanoemulsion gel as a function of oscillation stress. (* indicates p < 0.05).
CE. However, in our experimental design, we were unable to fully examine the effect of further increasing the viscosity through the addition of PF-127. To elucidate the effect of the gelling agent on system stability, an even more severe storage condition, such as freezing and melting, might need to be included in the accelerated test. Nevertheless, based on our results and previous literature, we expect that storage life will be much longer for the gelled formulation than the liquid formulations (Heurtault, Saulnier, Pech, Proust, & Benoit, 2003; Mou et al., 2008).
time, but a homogenized application will be more difficult.
3.6. Factors that influence storage stability To investigate the factors that influence storage stability, the accelerated storage test was applied to CE, NE, and NG samples. The effect of droplet size was clearly demonstrated when the CE developed an unstable 2-phase structure while the NE emulsion remained a homogenized mixture (Fig. 4). Though all behaved as shear-thining fluids, the viscosity of the prepared formulation was 0.35 ± 0.01, 0.63 ± 0.07, 9.48 ± 0.3, 12.27 ± 0.22, 14.30 ± 0.21 Pa s for CE, NE, and NG containing 18%, 20, and 22% of PF-127, respectively (Table 3). According to Stoke's law, a higher viscosity should contribute more to the stability of the system. This rule is observed as the more viscous NE system showed better storage stability than the less viscous
3.7. Percutaneous absorption The effect of topical formulations on the percutaneous absorption rate of the bioactive component in ABO was evaluated in vitro using a Franz diffusion cell. The skin permeability of bioactive components was
Fig. 4. Physical appearance of (A) emulsion, (B) nanoemulsion and (C) nanoemulsion gel at 0 and 10 days of accelerated storage study. The ratio of water, adlay bran oil and Tween 80 in the emulsion and nanoemulsion was 60:28:12%. Nanoemulsion gel contained 18% PF-127. 6
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Table 3 Viscosity of different formulations at 37 °C. Emulsion
Viscosity (Pa·s)
0.4 ± 0.0
Nanoemulsion
Nanoemulsion gel
0.6 ± 0.1
(18% PF-127)
(20% PF-127)
(22% PF-127)
9.5 ± 0.3
12.3 ± 0.2
14.3 ± 0.2
*Shear rate = 100 Pa/s. The results are expressed as the mean ± SD (n = 3).
Fig. 5. Percutaneous absorption rate of (A) different dosage forms and (B) nanoemulsion gel containing 18%, 20%, and 22% of PF-127 through the Franz diffusion cells. * Indicates significant difference from the unprocessed oil group. ** Indicates significant difference from all other experimental groups.
for the design of an effective nanoemulgel system for topical uses.
monitored by quantifying the amounts of phenolic compounds in the receiver chamber through time. The permeation profile of bioactive components through pig skin is shown in Fig. 5. The crude ABO, unsurprisingly, showed the lowest permeation rate among all applied dosages. Transdermal permeation of a bioactive component is determined by its diffusion and release rate from the vehicle as well as its permeability through the skin (Peltola, Saarinen-Savolainen, Kiesvaara, Suhonen, & Urtti, 2003). Therefore, the NE was expected to be the most effective system (Fig. 5A) for enhancing permeability since it promotes both good compound mobility within the system and a higher rate of cellular uptake (Mou et al., 2008). Although it may also allows better compound mobility, the CE with larger droplets was less effective than NE to facilitate compound permeability since larger droplet size is disadvantageous for the compound uptake by percutaneous cells. Similar result was found in many other previous reports where smaller emulsion droplet size would significantly increase the celllular uptake as well as permeation rate of bioactive components(Li, Nielsen, & Müllertz, 2016; Otto, Du Plessis, & Wiechers, 2009; Sonavane et al., 2008). Unlike transdermal delivery, a topical application requires that the active component accumulates on the skin and with minimal permeation (Escobar-Chávez et al., 2006). The inclusion of PF-127 to the NE system decreased the skin permeation rate of ABO bioactives (Fig. 5A). The higher viscosity of NG significantly reduced the compound mobility and, thus, increased the retention time on the skin surface. According to previous research works, improved local retention would enhance the efficacy while reducing the systemic toxicity of bioactive component (Lin et al., 2014; Sharma & Pathak, 2011). In this sense, NG is then a superior dosage form than the liquid emulsion formulation for topical application, which would benefit from higher accumulating rate of active compounds. As NG containing 18%, 20%, and 22% of gelator showed similar permeation profiles, the concentration of PF-127 has found not significantly affect the percutaneous absorption (Fig. 5B). This observation indicated that inclusion of PF-127, even at lower concentration, would be sufficient to reduce compound mobility and facilitate necessary compound retention on the skin surface. Therefore, using PF-127 at 18% concentration an optimal and economical choice
4. Conclusion We have successfully developed an NG system for topical application. The factors that influence the physical and chemical properties of the product were elucidated. Operational parameters of high-pressure homogenization were investigated to optimize the production process. In addition, the changes in formulation stability due to smaller droplet size and higher viscosity were also examined. Overall, the addition of a gelator, PF-127, to the NE system increased the system viscosity, thus decreasing the mobility of the bioactive component and regulating its release kinetics, which led to a lower skin permeation rate and, presumably, a higher level of cellular accumulation. Therefore, the NG system is a favorable vehicle for topical application, whereas the NE system is advantageous for the transdermal delivery of bioactives. The techniques and analytical methods used in this study will be a useful reference for the future development of suitable dosages for various delivery routes.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Conflict of interest file There was no conflict of interest to disclose.
Acknowledgments This work is supported by the Ministry of Science and Technology, Republic of China (Grant No. 107-2320-B-002-003-MY3 and No. 1052320-B-002-004-). 7
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Development of a topical applied functional food formulation: Adlay bran oil nanoemulgel
T
Wen-Chang Changa,b,1, Yin-Ting Hua,1, Qingrong Huangc, Shu-Chen Hsieha,∗∗, Yuwen Tinga,∗ a
Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City, Taiwan Department of Food Science, National Chiayi University, No. 300, Syuefu Rd., Chiayi City, Taiwan c Food Science Department, Rutgers University, 65 Dudley Rd., New Brunswick, NJ, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adlay bran oil Functional food By product Topical delivery Emulgel
Using bioactive ingredient from natural food sources to treat chronic health problems has become an emerging trend. Compare to oral consumption, topical application is still the preferred route for administrating these active food ingredients to skin for direct treatment of dermal diseases. Emulgel, a polymeric emulsion system, possesses the properties of both emulsions and hydrogel. Thus, a polymeric emulsion dosage could encapsulate both hydrophilic and hydrophobic compounds and improves their storage stability and cellular uptake. Extracted from the by-product of adlay processing, adlay bran oil is found to ameliorate many skin related diseases. Through the construction of a pseudo-ternary phase diagram, a formulation that contains 60% water, 28% adlay bran oil, and 12% Tween 80 was selected since it generates nano-droplets while still maintaining a sufficient surface energy for better stability. Two cycles of 1000 bar pressure treatment were required to produce nanoemulsion using high-pressure homogenizer. The addition of Pluronic® F-127 successfully transformed nanoemulsion into nanoemulgel. Depending on the gelator concentration, the sol-gel transition temperature varies from 20 to 30 °C. Apart from nanoemulsion facilitating higher permeation rate, nanoemulgel promoted the accumulation of bioactive component on the skin is a more suitable functional food formulation for topical uses.
1. Introduction Adlay bran oil (ABO), obtained from pressing the bran of adlay, or Job's tears (Coix lachrymal-jobi L. var.ma-yuen Stapf), is the main byproduct of the adlay refining process. It has good antioxidant, anti-inflammatory, anti-pigmentation, and anti-cancer effects due to its high content of bioactive components (C.-J. Huang et al., 2015). The phytochemical constituents isolated from adlay bran can be divided into nine main categories—namely, phenolic acids, phenolic esters, phenolic aldehydes, flavonoids, triterpenoids, sterols, lactams, lactones, and lignans. (C.-J. Huang et al., 2015). Using ABO to develop health-promoting formulations is desirable because it reduces production waste and increases the economic value of adlay processing. As the largest organ of the human body, the skin is the most critical barrier involved in preventing the invasion of pathogens as well regulating body temperature. However, the skin is vulnerable to skin cancer, one of the most common cancers worldwide, as well as other diseases. The use of emulsions can enhance the efficacy of bioactive
ingredients used to treat the skin diseases. According to previous research, an emulsion is better absorbed when it is processed to a smaller size and is allowed to stay in contact with the skin longer (Pouton, 1985). Therefore, it is desirable to reduce the size of the emulsion droplets and to increase surface retention time when developing emulsion products for skin use. The formation of an emulsion requires the intervention of an emulsifier to facilitate the uniform dispersion of two immiscible liquids. Selecting a suitable emulsifier can promote the formation of a homogeneous system with better storage stability (Alexander et al., 2013). The stability of an emulsion can be extended by processing it into a gelled system by incorporating a gelling agent (Mohamed, 2004). In this sense, oil-in-water (O/W) or water-in-oil (W/O) emulsions form a macromolecular network structure that prevents the aggregation of the dispersed phase and, thus, improves the stability of the continuous phase. An emulgel is an emulsion mixed with a gelling agent and it is a favorable delivery system for bioactive ingredients when a longer retention time is required (Devada, Jain, Vyas, & Jain, 2011).
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (S.-C. Hsieh), [email protected] (Y. Ting). 1 These authors contribute equally. ∗∗
https://doi.org/10.1016/j.lwt.2019.108619 Received 14 February 2019; Received in revised form 9 September 2019; Accepted 10 September 2019 Available online 11 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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Topical administration using delivery systems such as emulsions, ointments, and hydrogels is recommended for many skin problems. However, these topical dosages all have one or more drawbacks, including poor loading concentration, low diffusion coefficient, insufficient stability, and their limitation to the delivery of only hydrophiles or lipophiles. To enhance the functionality of an available topical delivery system, a two-step process that transforms the liquid emulsion into a semisolid emulsion gel has been developed. Emulgel, a polymeric emulsion system, has the advantages of both an emulsion and a hydrogel system. With the proper design, an emulgel system with a shear thinning property could reproduce not only the excellent stability of a hydrogel, but also the superior skin permeability of liquid emulsions (Devada et al., 2011). The wide variety of gelling agents that are soluble in either oil or aqueous environments allow the encapsulation of both hydrophobic and hydrophilic components in the O/W and W/O emulsions, respectively. Moreover, an emulgel that has a higher viscosity than the liquid emulsion system can increase the retention time of active components on the skin surface and promote better absorption (Mohamed, 2004). Overall, emulgels are a favorable formulation in the percutaneous absorption of both hydrophilic and lipophilic components because they offer alternative phases (dispersed and a continuous), higher skin permeability, better application and storage stability, and the ability to control the release rate. As bioactives isolated from adlay are effective against many skinrelated problems, it is rational and economical to use the oil extracted from adlay bran for skin product development. Specifically, adlay bioactives have been documented to be effective in inhibiting cellular tyrosinase activity and melanin production and, thus, preventing hyperpigmentation, which results from hormonal changes, aging, skin diseases, or injuries (H.-C. Huang, Hsieh, Niu, & Chang, 2014). In this study, an emulsion-based delivery system, including a conventional emulsion (CE) (d > 1 μm), nanoemulsion (NE) (d < 500 nm) (Chime, Kenechukwu, & Attama, 2014; Qian, Decker, Xiao, & McClements, 2012; Zhou, Wang, Cheung, Guo, & Jia, 2008), and nanoemulgel (NG), was prepared and optimized for skin use. The gelling agent selected for the preparation of nanoemulgel system was Pluronic F-127 (PF-127), which is one of the poloxamer ABA block polymer. The special chemical structure of PF-127 makes it more readily soluble in cold than in hot water and, thus, would allow it to maintain a gel-like property at body temperature. The physicochemical properties and percutaneous absorption rate of the prepared skin formulations were then studied systematically. This study is the first to report the production of an emulsion system using ABO. Our results not only demonstrate the potential to re-use a manufacturing by-product but also serve as an excellent reference for selecting a different delivery system for topical applications.
ABO was simultaneously filtered, collected using a serum bottle and stored in the dark at 4 °C until future application. The oil extraction yield was approximately 20%.
2. Materials and methods
To avoid multiple scattering effects, samples were diluted 500-fold with DI water before measurement. After dilution, the samples were analyzed by the dynamic light scattering method (Nanotrac 150, Microtrac, Montgomeryville, PA, USA). The droplet size (nm) was estimated as the mean diameter (in microns) of the volume distribution from 3 replicates:
2.3. Construction of pseudo-ternary phase diagram The pseudo-ternary phase diagram of the ABO, Tween 80®, and deionized water was constructed using a method reported by Xi et al. with minor modifications (Xi et al., 2009). Briefly, the oil phase was prepared by mixing ABO and Tween 80 at weight ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. The prepared oil phase was then combined with deionized (DI) water by the aqueous titration method, in which 100 μL of DI water was gradually added each time and the proportion of DI water was progressively increased. After homogenizing for 2 min with a high-speed homogenizer (T25 digital, IKA, Germany) at 25,000 rpm, samples were left standing in the dark for 1 h at room temperature (~25 °C). After 1 h, the physical status of each emulsion sample was observed and recorded.
2.4. Preparation of emulsion and nanoemulsion (NE) samples To prepare the emulsion system, ABO and Tween 80 were added to the glass bottle at a weight ratio of 9:1, 8:2, 7:3, and 6:4, mixed well and, combined with DI water. The conventional emulsion (CE) was prepared by homogenizing the mixture for 2 min with a high-speed homogenizer (T25 digital, IKA, Germany) at 25,000 rpm. To produce a nanoemulsion, the emulsion was subjected to a high-pressure homogenizer (N-2 Nanolyzer, Gogene Co., Taiwan) at a pressure of 1000 bar. To facilitate observation, ABO was first combined with 1% (v/v) Oil red O stain before preparing the CE and NE samples used for the construction of pseudo-ternary phase diagram and the study of accelerated storage stability.
2.5. Preparation of nanoemulgels (NGs) Based on the result obtained from the pseudo-ternary diagram, the optimum NE with the smallest droplet size was selected for further preparation of the NG. The gelling agent (Pluronic® F-127) was first dissolved in DI water and then mixed with NE at final concentration of 10%, 12%, 15%, 18%, 20%, and 22% in a glass bottle. The gel structure appeared after being stirred on a magnatic stirr plate at 4 °C for 24 h.
2.6. Droplets size analysis
2.1. Chemical reagents Folin-Ciocalteu phenol reagent and sodium carbonate were purchased from Merck (Darmstadt, Germany). Pluronic® F-127 (PF-127), Tween® 80 and Oil red O were obtained from Sigma-Aldrich (St. Louis, MO, USA). Kojic acid was purchased from TCI (Tokyo, Japan). All other chemicals and solvents were of analytical grade. Deionized water was prepared with a Milli-Q water purification system (Millipore, USA).
MV = ΣVidi / ΣVi
(1)
where MV is the mean volume diameter (μm), V is the volume percent (%) of measured droplet size and d is the droplet diameter (μm). All results are present as the average measurement of samples produced from 3 separate batches.
2.2. Preparation of adlay bran oil (ABO) Adlay (Taichung selective No. 4) cultivated and taxonomically identified by the Taichung District Agricultural Improvement Station (Taichung, Taiwan) was selected for the experiment. The oil was obtained by pressing only the adlay bran (the outer shell) with a lowtemperature presser (SP-230,SHANQ JER industries CO. Ltd., Taiwan). The adlay bran was packed in a filter cloth and pressed in two stages.
2.7. Zeta potential analysis Each sample was diluted 500-fold with DI water before being subjected to zeta potential analysis (Zetasizer Nano Z, Malvern Instruments Co., Ltd., Worcestershire, United Kingdom). 2
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collected from the receiving chamber of the Franz diffusion cell was analyzed by a microplate reader (Epoch™ Microplate Spectrophotometer, BioTek Instruments, Winooski, Vermont, USA) as reported by Singleton et al. (Singleton & Rossi, 1965). The standard curve was constructed using 0, 0.625, 1.25, 2.5, 5, 10, 25, and 50 μg/ mL of gallic acid in a volume of a 25 μL per well in a 96-well plate. Later, 25 μL of Folin-Ciocalteu reagent was added to each well and allowed to react in the dark for 5 min, then 50 μL of DI water and 150 μL of 5% Na2CO3 solution were added to each well and again incubated for 1 h in dark. The reader then determined the light absorption value at 750 nm. The total phenolic content in the sample was determined from the standard curve and expressed in terms of gallic acid equivalent concentration (μg/mL).
2.8. Rheological analysis A rheometer (AR 2000ex, TA Instruments, New Castle, DE, USA) was used to measure the rheological behavior of the prepared samples. Sample viscosity was measured using a 40 mm diameter parallel plate set at a 2 mm gap distance at 25 °C. Data were recorded every 5 s for a total of 2 min, during which angular frequency ω range from 1 to 200 rad/s was gradually applied to the sample. In the stress sweep test, changes on the storage modulus (G′) were studied when 0.1–1000 Pa of the oscillation stress was applied to samples at a fixed frequency of 1 Hz. Finally, the gelling behavior of NG over a temperature ramp was also determined through a temperature increase from 4 to 80 °C at a frequency of 1 Hz and 0.5% strain. Values of storage modulus (G′) and loss modulus (G″) were obtained every 30 s and Tan δ was calculated accordingly. The rate of temperature increase and the rate of temperature drop were both 1 °C per minute.
2.12. Statistical analysis All tests were conducted in at least 3 replicates and were presented as the average of measurements. The data were analyzed using a oneway analysis of variance and Duncan's new multiple-range test using SAS statistical software package (SAS Institute, Cary, NC, USA). P values of < 0.05 were considered significant. The data are shown as the mean ± standard deviation (SD).
2.9. Accelerated storage test Storage stability is an important quality parameter commonly studied by investigators when developing new formulations. Instead of the real-time stability test, the accelerated test using elevated stress conditions is more commonly applied to rapidly predict the degradation rate of candidate formulations. The test we used was carried out with a slight modification based on the method of Restu et al. and Smrity et al. (Restu, Sampora, Meliana, & Haryono, 2015; Smrity, Saifuddin, & Sultana, 2016). To complete one accelerated storage cycle, an equal amount of CE, NE, and NG were put into separate glass bottles, capped, refrigerated at 4 °C for 24 h, and then moved to a 45 °C chamber to store for another day. The sample used for the accelerated storage test were observed for any sign of physical changes. In addition, the droplet size and zeta potential of all samples were determined at the end of each of five temperature cycles. The sample was considered stable if it remained physically unchanged and maintained constant droplet size and surface charges.
3. Results and discussion 3.1. Construction of pseudo-ternary phase diagram By constructing a pseudo-ternary phase diagram, all possible mixing ratios of the emulsifier and the organic and aqueous phases were studied systematically, resulting in the elucidation of formulations of various physicochemical properties. Using a titration method, we investigated the existence of a stable emulsion region where the organic phase was composed of 10–90% of the emulsifier (Tween 80) in ABO and was titrated by the aqueous phase (DI water) at various ratios (Fig. 1A). Starting at 9-to-1 ratios of organic-to-aqueous phases, stable water-in-oil (W/O) emulsions were formed when the organic phase contained more than 60% of Tween 80. As more of the water was added, the emulsion systems underwent a liquid-to-semisolid transition, during which gel-like structures were formed (indicated by the solid black circle in the diagram). Later, the semi-solid formulations experienced another transition back to the liquid form, when the percentage of the aqueous phase was increased within the range of 35–60% (w/w) in the systems containing lower to a higher proportion of emulsifier, respectively. The result is consistent with previously published results, describing a linear correlation between concentration and emulsification power, which was indicated by more stable emulsion formulations that were formed with higher contents of Tween 80 (Chen & Tao, 2005; Fernandez, André, Rieger, & Kühnle, 2004). To produce droplets in the nanoscopic range, emulsion systems should have the suitable flow property enabling them to pass through the high energy delivering instrument, such as a high-pressure homogenizer. The flow property (commonly known as viscosity) of an emulsion system is inversely correlated to the oil content of the formulation (Floury, Desrumaux, & Lardieres, 2000). However, in the case of ABO as the organic phase, it is desirable to have a higher oil content, because of all the bioactive components contained within. Therefore, it is desirable to maximize the oil content in the emulsion while still allowing its smooth passage through the high-energy homogenizer. The semi-solid formulations, although containing a higher amount of oil, have poor fluidity and, thus, were not selected for further processing into a nanoemulsion. According to the phase diagram, all formulations containing more than 60% water had liquid-like fluidity regardless of the emulsifier content in its organic phase. Tween 80 is simultaneously an emulsifying and gelling agent, thus its concentration significantly influences the viscosity of a formulation (Prieto & Calvo, 2013; Sagiri, Behera, Sudheep, & Pal, 2012). In Fig. 1B,
2.10. Percutaneous absorption test The skin absorption rate was determined in vitro using a Franz diffusion cell. Briefly, the effective absorption area of the device was first covered by a 6.25 cm2 piece of back pigskin (0.3–0.5 mm thick) and then assembled with an upper and a lower chamber using a metal clamp. The skin from a 3-4-month-old pathogen-free miniature pig was directly purchased from the Agricultural Technology Research Institute (Taipei, Taiwan). Approximately 0.5 g of the formulation prepared for skin absorption was placed in the upper chamber. At 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h, 500 μL of sample was collected from the lower receiving chamber, which was initially filled with 11 mL of PBS. The concentrations of phenolic compounds from the collected samples were analyzed using a spectrophotometric method that will be described in a later section. Compound penetration volume per unit time was calculated using the following formula: Wn (μg) = Cn × V + Vs × (C1 + C2 + C3 + … …..Cn-1)
(2)
where Wn is the weight of the total phenolic compounds in the receptor fluid during the nth sampling, Cn is the concentration of the total polyphenolic compounds in the receptor fluid during the nth sampling, V is the total volume of the receptor fluid, and Vs is the volume of the receptor fluid during each sampling. Finally, the cumulative compound penetration volumes were plotted against time to study the absorption kinetics. All experiments were repeated at least three times (n = 3). 2.11. Total phenolic contents The concentration of the phenolic compound in the samples 3
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Fig. 1. (A) Pseudo-ternary phase diagram and (B) viscosity of the emulsion composed of adlay bran oil, Tween 80, and water (60%). The results are expressed as the mean ± SD of each group (n = 3).
Despite the fact that a nonionic emulsifier was used, the surface of the emulsion droplet was found to be negatively charged due to the spontaneous charging of the oil-water interface, to which hydroxyl ions released by the dissociated water molecules were absorbed (Marinova et al., 1996). This phenomenon is supported by the negative zeta potential values that were detected on the surface of all formations (Table 1). However, the concentration of emulsifier did not significantly affect the magnitude of the absolute surface charge until the droplet size was further reduced to less than 300 nm by a high-pressure homogenizer. The resulting increase in surface area covered with uncharged emulsifier led to a reduction in the number of absorption sites for the hydroxyl molecule, thus lowering the magnitude of the surface charges. That is, high-pressure homogenization not only effectively reduced the droplet size but also significantly decreased the amount of surface charges on the droplets as indicated by the zeta potential measurement. All formulations except for the one that contained 40% Tween 80 in the organic phase maintained its zeta-potential value of more than (−) 30 mV. This observation could be attributed to the excessive surfactant absorption on the droplet surface forming a thick protection layer that prevented the hydrogen bonding between the absorbed hydroxyl group and a water molecule. As stated previously, if the absolute magnitude of the zeta potential is higher than 30 mV, then it can contribute to the stability of the colloidal system (Patel & Agrawal, 2011). Thus, to enhance the systemic stability, it is desirable to select a formulation that can produce an emulsion of nanoscopicsized droplets and with a more substantial surface charge.
a positive relationship between the proportion of Tween 80 in the organic phase and viscosity of the formulation was shown when water content was kept constant at 60%. Since the formulations that could successfully pass through the available high-pressure homogenizer were only those with a viscosity of less than 1 Pa s, the oil phase of the emulsion system could not contain more than 40% of the Tween 80. 3.2. Properties of the emulsion (CE) and nanoemulsion (NE) To find the optimum mixing ratio, the physical properties of formulations containing 60% aqueous phase and 40% organic phase were analyzed. Based on the rheological analysis, formulations with an organic phase containing 10%–40% of Tween 80 in the ABO could be further processed using a high-pressure homogenizer. As indicated by Stoke's law, the rate at which an emulsion system becomes unstable is positively correlated with its droplet size. After subjecting the system to high-speed homogenization, the droplet size of formulations containing 10%, 20%, 30%, and 40% of Tween 80 in the oil phase were approximately 1345.50, 951.00, 610.50, 590.00 nm, respectively. This result agrees with a previous investigation where the ratio of grease to Tween 80 had a significant effect on the droplet diameter of the emulsion system (Chen & Tao, 2005). After subjecting the system to high-pressure homogenization, the droplet size of all formulations decreased by 4-12-fold, depending on the concentration of the emulsifier. However, only formulations containing 30% and 40% of Tween 80 in the organic phase contained droplets with an average size of less than 100 nm (Table 1), which is a strict requirement for the nanoscopic product (Bhattacharjee, 2016).
3.3. Effect of homogenization parameters on the droplet diameter Table 1 Physical properties of adlay oil emulsion and nanoemulsion with different adlay oil and Tween 80 ratios. Properties D (nm)
ζ (mV)
Percentage of Tween 80 (%) 10 20 30 40 10 20 30 40
Emulsion 1345.5 ± 246.9 951.0 ± 27.3b 610.5 ± 68.8c 590.0 ± 110.0c −48.4 ± 0.8a −44.2 ± 1.1b −46.3 ± 1.7ab −40.3 ± 0.8c
The size of the emulsion droplet is affected by the processing parameters during high-pressure homogenization. Among all influencing factors, the level of applied pressure and number of input cycles most significantly affect droplet diameter (Qian & McClements, 2011). Based on previous section results, a formulation containing 60% DI water, 28% ABO, and 12% Tween 80 was chosen to manufacture NE. The selected formulation was subjected to processing pressures of 500, 1000, and 1500 bars for 1–5 cycles. After the first cycle, the emulsion droplet size was significantly reduced under all pressure magnitudes. Comparing the three processing conditions, an applied pressure of 500 bar was less efficient since it produced larger droplets than applied pressures of 1000 and 1500 bars at all processing cycles (Table 2). Although the 1500 bar pressure could produce the smallest emulsion droplets within the 1st cycle, it also generated excessive heat that caused rapid water evaporation and, thus, increased the chance of
Nanoemulsion a
214.4 ± 14.0a 197.1 ± 14.1b 73.7 ± 9.0c 46.0 ± 2.3d −34.2 ± 1.2a −36.3 ± 1.2a −33.0 ± 2.8a −25.8 ± 0.9b
The results are expressed as the mean ± SD of each group (n = 3). Values with different letters (a-d) between columns indicate a statistically significant difference among emulsion and nanoemulsion (p < 0.05). D is particle size, ζ is zeta-potential, and μ is viscosity at 100 s−1 of shear rate. 4
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Table 2 Effect of processing pressure and cycles on the droplet diameter. Droplet diameter (nm) Pressure (bar) 500 1000 1500
Cycle 1 127.8 ± 8.0a 78.1 ± 3.4a 66.9 ± 6.8a
Cycle 2 94.8 ± 5.1b 61.6 ± 4.2b 59.7 ± 5.2a
Cycle 3 83.0 ± 4.3cd 61.1 ± 1.4b 61.2 ± 3.4a
Cycle 4 90.3 ± 3.3bc 60.5 ± 2.7b 61.0 ± 4.8a
Cycle 5 77.2 ± 3.8d 63.8 ± 2.3b 66.5 ± 1.8a
The results are expressed as the mean ± SD of each group (n = 4). Values with different letters (a-d) indicate a statistically significant difference (p < 0.05) between the number of cycles under 500, 1000, or 1500 bar pressures.
capacity than other carrier systems (Mou et al., 2008). In the specific case of topical application, sufficient formulation viscosity is necessary for sufficient skin contact time, during which the bioactive compounds contained within can be released and permeate into the skin (Bhattacharjee, 2016). Thus, a suitable polymeric gelling agent is usually added to the liquid formulation to enhance its stability and skin retention time by modifying its rheological property (Valenta & Schultz, 2004). Pluronic F-127 (PF-127), a non-ionic gelling agent, forms monomolecular micelles and multimolecular aggregates (micelles ordered into a lattice) at low and high concentrations, respectively. Thus, a higher concentration (> 15%) of PF-127 is required for the construction of the gel structure (Patel & Agrawal, 2011). In this investigation, the emulsion system was only starting to gel when the concentration of PF-127 was higher than 18% (w/w). A similar phenomenon was reported by Nie et al., who showed that 18% of PF-127 is required for the preparation of a liposomal gel (Hsu & Nacu, 2003). Besides concentration, the gelation property of PF-127 is also affected by temperature. An aqueous solution containing 20–30% (w/w) of PF-127 shows an interesting reversible thermal gelation behavior (Lenaerts, Triqueneaux, Quartern, Rieg-Falson, & Couvreur, 1987; Miyazaki, Takeuchi, Yokouchi, & Takada, 1984), i.e., the solution is liquid at a refrigerated temperature and will slowly gel as the temperature is increased. The temperature at which the PF-127-containing formulations change from the liquid state to a gel state is significantly influenced by the concentration. The sol-gel transition occurred when the temperature was increased to 30.6 ± 0.3, 25.5 ± 0.6, and 20.5 ± 0.1 °C for formulations containing 18%, 20% or 22% of PF-127, respectively (Fig. 3A). The experimental data supports the inverse relationship between the PF-127 concentration and the sol-gel transition temperature. Thus, thermally reversible properties were also observed, as would be expected from the results of a previous investigation (Liu, Sun, Li, Liu, & Xu, 2006). The formation of a polymer micelle is mainly driven by the hydrophobic interaction between the polyoxyethylene-polyoxypropylene copolymers in PF-127 (Hunter, 2001). Thus, any factor that interferes with the hydrophobic interaction between PF-127 molecules will significantly decrease micelle formation and, thus, disturb the molecular arrangement into an ordered lattice structure (IWH, 1998.). Comparing PF-127 concentrations, the inclusion of NE in the system significantly raised the sol-gel transition temperature when the PF-127 concentration was at 18% and 20% (Fig. 3B). This phenomenon implies that PF127 can be absorbed onto the droplet surface and, thus, its efficient micelle aggregation is disturbed by the presence of an emulsion. When the temperature was further increased, more of the hydrophilic chains of copolymer became desolvated, which favors the hydrophobic interaction (Miller & Drabik, 1984). Thus, the addition of NE was less influential to the sol-gel transition temperature of PF-127 when its concentration was 22%. Spreadability, a fundamental rheological property, was studied by monitoring the change in the storage modulus (G′) when applying a sweep of shear stress to a topical dosage. As shown in Fig. 3C, the G′ value and the yield stress of NG went up as the PF-127 concentration increased. That is, the rigidity and strength of NG increased as the concentration of PF-127 increased. The result indicates that the formulation that contains more PF-127 can improve its skin retention
machine blockage due to higher sample viscosity. Under 1000-bar pressure, more than two runs through the homogenizer was necessary since the sample droplet size did not change significantly after the 2nd run (Table 2). Given the alternatives, we therefore selected an applied pressure of 1000 bars as the final processing pressure for high-pressure homogenization, even though it requires two processing cycles to generate satisfactory nanodroplets,. 3.4. Effect of high-pressure processing on storage stability To study the system stability, emulsion samples before and after high-pressure processing were studied using the accelerated storage test. Before high-pressure treatment, the CE (diameter > 500 nm) phase separated after being subjected to the accelerated test for one day, whereas the NE (diameter < 100 nm) maintained its stable 1phase structure even after ten days of storage under extreme conditions. During the experiment, there was no significant change in the droplet size of NE, further indicating good storage stability (Fig. 2). Results of the accelerated storage test show that high-pressure processing effectively reduces the droplet diameter of the emulsion, while at the same time enhancing the stability of the system. This result is consistent with Stoke's law, the droplet diameter is inversely related to its velocity of becoming unstable. More evidance on the enhance stability thorugh decreasing the droplet size was also found in previously published literatures (Abismaıl, Canselier, Wilhelm, Delmas, & Gourdon, 1999; Vladisavljević & Schubert, 2003). 3.5. Physical properties of adlay oil nanoemulgel Unlike microemulsions, an NE is a non-equilibrium system and will eventually become unstable after storage for an extended period of time. However, NE is commonly used in pharmaceutical and cosmetic products because it offers several significant advantages, such as lower skin irritation, better membrane permeability, and higher loading
Fig. 2. Particle size of adlay bran oil nanoemulsion measured during the accelerated storage test. 5
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Fig. 3. Storage modulus (G′) and loss modulus (G″) of a nanoemulsion gel containing (A-a) 18%, (A-b) 20%, and (A-c) 22% of PF-127 as a function of temperature. (B) Gelation temperature of the nanoemulsion gel contained various amount of PF-127. (C) Storage modulus (G′) of nanoemulsion gel as a function of oscillation stress. (* indicates p < 0.05).
CE. However, in our experimental design, we were unable to fully examine the effect of further increasing the viscosity through the addition of PF-127. To elucidate the effect of the gelling agent on system stability, an even more severe storage condition, such as freezing and melting, might need to be included in the accelerated test. Nevertheless, based on our results and previous literature, we expect that storage life will be much longer for the gelled formulation than the liquid formulations (Heurtault, Saulnier, Pech, Proust, & Benoit, 2003; Mou et al., 2008).
time, but a homogenized application will be more difficult.
3.6. Factors that influence storage stability To investigate the factors that influence storage stability, the accelerated storage test was applied to CE, NE, and NG samples. The effect of droplet size was clearly demonstrated when the CE developed an unstable 2-phase structure while the NE emulsion remained a homogenized mixture (Fig. 4). Though all behaved as shear-thining fluids, the viscosity of the prepared formulation was 0.35 ± 0.01, 0.63 ± 0.07, 9.48 ± 0.3, 12.27 ± 0.22, 14.30 ± 0.21 Pa s for CE, NE, and NG containing 18%, 20, and 22% of PF-127, respectively (Table 3). According to Stoke's law, a higher viscosity should contribute more to the stability of the system. This rule is observed as the more viscous NE system showed better storage stability than the less viscous
3.7. Percutaneous absorption The effect of topical formulations on the percutaneous absorption rate of the bioactive component in ABO was evaluated in vitro using a Franz diffusion cell. The skin permeability of bioactive components was
Fig. 4. Physical appearance of (A) emulsion, (B) nanoemulsion and (C) nanoemulsion gel at 0 and 10 days of accelerated storage study. The ratio of water, adlay bran oil and Tween 80 in the emulsion and nanoemulsion was 60:28:12%. Nanoemulsion gel contained 18% PF-127. 6
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Table 3 Viscosity of different formulations at 37 °C. Emulsion
Viscosity (Pa·s)
0.4 ± 0.0
Nanoemulsion
Nanoemulsion gel
0.6 ± 0.1
(18% PF-127)
(20% PF-127)
(22% PF-127)
9.5 ± 0.3
12.3 ± 0.2
14.3 ± 0.2
*Shear rate = 100 Pa/s. The results are expressed as the mean ± SD (n = 3).
Fig. 5. Percutaneous absorption rate of (A) different dosage forms and (B) nanoemulsion gel containing 18%, 20%, and 22% of PF-127 through the Franz diffusion cells. * Indicates significant difference from the unprocessed oil group. ** Indicates significant difference from all other experimental groups.
for the design of an effective nanoemulgel system for topical uses.
monitored by quantifying the amounts of phenolic compounds in the receiver chamber through time. The permeation profile of bioactive components through pig skin is shown in Fig. 5. The crude ABO, unsurprisingly, showed the lowest permeation rate among all applied dosages. Transdermal permeation of a bioactive component is determined by its diffusion and release rate from the vehicle as well as its permeability through the skin (Peltola, Saarinen-Savolainen, Kiesvaara, Suhonen, & Urtti, 2003). Therefore, the NE was expected to be the most effective system (Fig. 5A) for enhancing permeability since it promotes both good compound mobility within the system and a higher rate of cellular uptake (Mou et al., 2008). Although it may also allows better compound mobility, the CE with larger droplets was less effective than NE to facilitate compound permeability since larger droplet size is disadvantageous for the compound uptake by percutaneous cells. Similar result was found in many other previous reports where smaller emulsion droplet size would significantly increase the celllular uptake as well as permeation rate of bioactive components(Li, Nielsen, & Müllertz, 2016; Otto, Du Plessis, & Wiechers, 2009; Sonavane et al., 2008). Unlike transdermal delivery, a topical application requires that the active component accumulates on the skin and with minimal permeation (Escobar-Chávez et al., 2006). The inclusion of PF-127 to the NE system decreased the skin permeation rate of ABO bioactives (Fig. 5A). The higher viscosity of NG significantly reduced the compound mobility and, thus, increased the retention time on the skin surface. According to previous research works, improved local retention would enhance the efficacy while reducing the systemic toxicity of bioactive component (Lin et al., 2014; Sharma & Pathak, 2011). In this sense, NG is then a superior dosage form than the liquid emulsion formulation for topical application, which would benefit from higher accumulating rate of active compounds. As NG containing 18%, 20%, and 22% of gelator showed similar permeation profiles, the concentration of PF-127 has found not significantly affect the percutaneous absorption (Fig. 5B). This observation indicated that inclusion of PF-127, even at lower concentration, would be sufficient to reduce compound mobility and facilitate necessary compound retention on the skin surface. Therefore, using PF-127 at 18% concentration an optimal and economical choice
4. Conclusion We have successfully developed an NG system for topical application. The factors that influence the physical and chemical properties of the product were elucidated. Operational parameters of high-pressure homogenization were investigated to optimize the production process. In addition, the changes in formulation stability due to smaller droplet size and higher viscosity were also examined. Overall, the addition of a gelator, PF-127, to the NE system increased the system viscosity, thus decreasing the mobility of the bioactive component and regulating its release kinetics, which led to a lower skin permeation rate and, presumably, a higher level of cellular accumulation. Therefore, the NG system is a favorable vehicle for topical application, whereas the NE system is advantageous for the transdermal delivery of bioactives. The techniques and analytical methods used in this study will be a useful reference for the future development of suitable dosages for various delivery routes.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Conflict of interest file There was no conflict of interest to disclose.
Acknowledgments This work is supported by the Ministry of Science and Technology, Republic of China (Grant No. 107-2320-B-002-003-MY3 and No. 1052320-B-002-004-). 7
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