Research and Development Division, Glico Nutrition Co., Ltd. (4-6-5 Utajima, Nishiyodogawa-ku, Osaka , JAPAN) 3

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1 Journal of Oleo Science Copyright 2015 by Japan Oil Chemists Society doi : /jos.ess14229 O/W Nano-Emulsion Formation Using an Isothermal Low-Energy Emulsification Method in a Mixture of Polyglycerol Polyricinoleate and Hexaglycerol Monolaurate with Glycerol System Satoshi Wakisaka 1, 2, Takahisa Nishimura 2 and Shoichi Gohtani 3* 1 Department of Food Science, The United Graduate School of Agricultural Sciences, Ehime University (2393 Ikenobe, Miki-cho, Kagawa , JAPAN) 2 Research and Development Division, Glico Nutrition Co., Ltd. (4-6-5 Utajima, Nishiyodogawa-ku, Osaka , JAPAN) 3 Department of Applied Biological Science, Faculty of Agriculture, Kagawa University (2393 Ikenobe, Miki-cho, Kagawa , JAPAN) Abstract: We investigated how phase behavior changes by replacing water with glycerol in water/mixture of polyglycerol polyricinoleate (PGPR) and hexaglycerol monolaurate (HGML) /vegetable oil system, and studied the effect of glycerol on o/w nano-emulsion formation using an isothermal low-energy method. In the phase behavior study, the liquid crystalline phase (Lc) + the sponge phase (L 3 ) expanded toward lower surfactant concentration when water was replaced with glycerol in a system containing surfactant HLP (a mixture of PGPR and HGML). O/W nano-emulsions were formed by emulsification of samples in a region of Lc + L 3. In the glycerol/surfactant HLP/vegetable oil system, replacing water with glycerol was responsible for the expansion of a region containing Lc + L 3 toward lower surfactant concentration, and as a result, in the glycerol/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/w emulsions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller than that in the water/surfactant HLP/ vegetable oil system. Therefore, glycerol was confirmed to facilitate the preparation of nano-emulsions from a system of surfactant HLP. Moreover, in this study, we could prepare o/w nano-emulsions with a simple one-step addition of water at room temperature without using a stirrer. Thus, the present technique is highly valuable for applications in several industries. Key words: nano-emulsion, isothermal low-energy emulsification, sponge phase, liquid crystalline, glycerol 1 INTRODUCTION Nano-emulsions are emulsions whose droplet diameter typically falls in the range of nm 1. The size of the droplets in nano-emulsions is often much smaller than the wavelength of light d << λ ; therefore, nano-emulsions do not scatter light strongly, making them transparent or translucent 2. Nano-emulsions also exhibit significantly improved emulsion stability against creaming and flocculation due to the substantially reduced rates of gravitational separation and enhanced Brownian motion of oil droplets 3. Finally, nano-emulsion technology has been applied in fabricating encapsulating systems for functional compounds because it prevents degradation and improves bioavailability 4. In general, nano-emulsions can be achieved using either high-energy emulsification methods or low-energy emulsification methods. High-energy emulsification methods involve an intensive energy input using a high-shear stirrer, a high-pressure homogenizer or ultrasound generators 1. Alternatively, low-energy emulsification methods that utilize thermodynamic driving forces have been developed, enabling the formation of nano-emulsions with minimal energy input of mechanical energy There is interest in using lower energy techniques in the emulsion formation process because of economic benefits. Low-energy emulsification methods can be broadly categorized as either thermal or isothermal methods 11. Thermal methods rely on emulsion formation resulting from changes in surfactant * Correspondence to: Shoichi Gohtani, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kagawa , JAPAN gohtani@ag.kagawa-u.ac.jp Accepted December 19, 2014 (received for review October 10, 2014) Journal of Oleo Science ISSN print / ISSN online

2 S. Wakisaka T. Nishimura and S. Gohtani properties with temperature, whereas isothermal methods rely on emulsion formation resulting from changes in local system composition at a fixed temperature 12. Spontaneous emulsification and phase inversion composition methods fall into the category of isothermal methods 13, while the phase inversion temperature method is an example of a thermal method 14, 15. In our previous study, we have demonstrated that nanoemulsions suitable for use in food systems can be prepared using an isothermal low-energy emulsification method, consisting of stepwise addition of water to the phase of the water/mixture of polyglycerol polyricinoleate PGPR and polyglycerol fatty acid ester PGFA /vegetable oil at a constant temperature 25 with moderate mixing using a stirrer operated at a rotational speed around 300 rpm 16. In comparing the phase diagram of the ternary system and the droplet size in the resulting emulsions, it was found that o/w nano-emulsions with droplet sizes as small as 50 nm were formed by emulsifying from either a single sponge phase L 3 or a two-phase region, liquid crystalline phase Lc L 3. These results indicate that L 3 or Lc, or both, is necessary to form an o/w nano-emulsion for PGPR and PGFA mixtures used as surfactant. Therefore, it can be expected that finding specific conditions to expand L 3 or Lc in the phase diagram for PGPR and PGFA mixtures may be applied to prepare an o/w nano-emulsion using an isothermal lower-energy emulsification method. It is known that the presence of cosolvents such as alcohols and polyols affect the phase behavior of surfactants dispersed in aqueous solutions and hence the outcome of low-energy emulsification is also affected 6, Among the cosolvents, glycerol is mostly used in industries such as pharmaceutical, cosmetic, food and chemical. Adding glycerol to aqueous solutions has been shown to alter the interfacial tension, optimum curvature and solubility characteristics of both ionic and non-ionic sufactants A number of studies have investigated the influence of glycerol and similar cosolvents on various types of emulsion-based systems 19, 20, 24. Suzuki et al. showed that the presence of an appropriate amount of glycerol enhanced the rigidity of bilayers forming a lamellar liquid crystal in an L-arginine hexyldecyl phosphate/water/glycerol system, thus promoting the formation of an oil-in-liquid crystal emulsion 5. As a result, they developed a liquid crystal emulsification technique known as the low-energy emulsification method. However, the effects of glycerol on a mixture of PGPR and PGFA systems have not yet been investigated. The objective of this research is to investigate how phase behavior changes by replacing water with glycerol in water/ mixture of PGPR and hexaglycerol monolaurate HGML / vegetable oil systems, and to determine the effect of incorporating glycerol in mixtures of PGPR and HGML on nanoemulsion formation using an isothermal low-energy method. In addition, unlike in our previous study where we added water step by step to the prepared phase while stirring 16, in this study, we attempted to prepare nano-emulsions with a simple one-step addition of water to the prepared phase without using a stirrer. 2 EXPERIMENTAL PROCEDURES 2.1 Materials PGPR commercial name SY-Glyster CRS-75, HGML commercial name SY-Glyster ML-500 were purchased from Sakamoto Yakuhin Kogyo Co., Ltd. Glycerol was purchased from Wako Pure Chemical Industries, Ltd. Vegetable oil consisting of soybean oil and rapeseed oil produced by Nisshin Oillio group Ltd. was purchased from conventional supermarket. All materials were used without further purification. Purified water was prepared using an E-pure system Dubuque, USA. We used a mixture of PGPR with HGML weight ratio 1:1 in this study. The mixtures of PGPR and HGML are abbreviated as surfactant HLP. 2.2 Methods Determination of the phase diagram Test tubes containing mixtures with the desired compositions were repeatedly shaken using a vortex mixer and then stored at 25. The formation of liquid crystals was confirmed using polarizing plates placed on either side of the test tube. If the liquid crystalline phase was formed, light passed through the plates due to the birefringence of the liquid crystalline. Detailed phases were identified using polarized light microscopy OLYMPUS, BH-2 and small-angle X-ray scattering SAXS techniques Small-angle X-ray scattering SAXS measurements were performed on a Nano-Viewer SAXS instrument Rigaku Co., Tokyo, Japan equipped with a PILATUS at an applied voltage and filament current of 40 kv and 30 ma, respectively. The wavelength of the radiation source was nm Preparation of an O/W emulsion Vegetable oil, water or glycerol and the emulsifier mixtures previously described in section 2.1 were mixed at the desired weight ratios to prepare mixtures before emulsification. The mixtures before emulsification were put in test tubes, and water was added to the test tubes in one step such that the final aqueous phase concentration was 99 wt. The test tubes were then gently shaken by hand at 25 to prepare an o/w emulsion Measurement of the particle size distribution and polydispersity Particle size distribution and polydispersity index PDI were determined using a dynamic light scattering device FPER-1000; Otsuka Electronics, Osaka, Japan. PDI provides a measure of the narrowness of the particle size dis- 406

3 Formation of O/W Nano-Emulsion Using Low-Energy Method with Glycerol tribution, with values << 0.1 indicating a very narrow distribution RESULTS AND DISCUSSION 3.1 Phase behavior To investigate how phase behavior changes by replacing water with glycerol in water/mixture of PGPR and PGFA/ vegetable oil system, phase diagram of the glycerol/surfactant HLP/vegetable oil system were determined. Figure 1 shows the phase diagram of the water/surfactant HLP/vegetable oil system A and the glycerol/surfactant HLP/vegetable oil system B at 25. Figure 1A is constructed based on previous article 16 with additional investigations to clearly explain differences between the system with water and the system with glycerol. For the water/surfactant HLP/vegetable oil system Fig. 1A, the two-phase region consisting of Lc L 3 or lamellar liquid crystal La L 3 was observed at surfactant HLP concentration above 35 wt. When the concentration of surfactant HLP was less than 35 wt, a three-phase region consisting of L 3 Wm O or Lc L 3 O, and a two-phase region consisting of Wm O appeared. Fig. 1 Phase diagram of (A) water/surfactant HLP/vegetable oil and (B) glycerol/surfactant HLP/vegetable oil systems at 25. Phase diagram (A) is constructed based on the previous article 16) with additional investigations. L 3, Lc, La, Wm, G and O indicate a sponge phase, a liquid crystalline phase, a lamellar phase, a micellar phase, a glycerol phase and an oil phase, respectively. In our previous study, we considered that the position where the concentration of surfactant HLP and water was below 35 wt% and below 20 wt%, respectively (dotted line in Fig. 1A), produced a two-phase region consisting of Lc + L 3 16). By additional investigations for the water/ surfactant HLP/vegetable oil system, we now presume the existence of a three-phase region consisting of Lc + L 3 + O as shown in Fig. 1A. 407

4 S. Wakisaka T. Nishimura and S. Gohtani Fig. 3 Photographs of samples represented by composition (a), (b) and (c) in Fig. 1B between crossed polarizes. Fig. 2 Polarized microscopic images and SAXS patterns of samples consisting of 25.0 wt%, 65.0 wt% and 10.0 wt% (a); 40.0 wt%, 20.0 wt% and 40.0 wt% (b); 70.0 wt%, 20.0 wt% and 10.0 wt% (c) of glycerol, surfactant HLP and vegetable oil, respectively. Compositions (a), (b) and (c) are indicated in Fig. 1B using the corresponding letters in parentheses. Figure 2 shows the polarized microphotographs, and SAXS patterns of mixtures of compositions a, b and c described in Fig. 1B and their visual states between crossed polarizes are indicated in Fig. 3. The sponge phase L 3 is often stated to be flow birefringent 25, and it is reported that the SAXS pattern obtained from L 3 indicates a broad peak 26. The scattering peak for the sample a in Fig. 1B was broad Fig. 2-a. On the other hand, this sample showed birefringence under polarization microscopy Fig. 2-a and between crossed polarizes in a static state Fig. 3-a. Consequently, sample a in Fig. 1B was identified as a two-phase coexisting with liquid crystalline Lc and L 3. As shown in Fig. 2, the SAXS pattern and the polarized microphotograph obtained from sample b in Fig. 1B showed a similar result to that of sample a. The SAXS pattern of sample c indicated a curved shape similar as a skirt of the broad peak of sample b shown in Fig. 2. We considered that the SAXS pattern obtained from sample c in Fig. 1B was the skirt of broad peak owing to L 3 Fig. 2-c. As shown in Fig. 3-b, c, oil or glycerol separated and the lower layer of sample b or the upper layer of sample c showed birefringence more intensively in flow state. This behavior is characteristic of L 3 phase as described in our previous report 16. These samples showed birefringence under polarization microscopy Fig.2-b, c, and showed birefringence in lower layer for sample b and between L 3 layer and glycerol layer for sample c as shown in Fig. 3. Consequently, sample b and c in Fig. 1B were determined as a three-phase coexisting with Lc, L 3 and oil phase Lc L 3 O, and Lc, L 3 and a glycerol phase Lc L 3 G, respectively as shown in Fig. 3. For the glycerol/surfactant HLP/vegetable oil system Fig. 1B, Lc L 3 was observed in all regions of the phase diagram. The two-phase region Lc L 3 appeared at surfactant HLP concentration above 35 wt. When surfactant HLP and the vegetable oil concentration was below 35 wt and below 20 wt, respectively, a three phase region with Lc L 3 G could be seen. When the vegetable oil concentration was increased to more than 20 wt at surfactant HLP concentration less than 35 wt, vegetable oil was gradually expelled from Lc L 3, resulting in the formation of a three phase region with Lc L 3 O or a four phase region with Lc L 3 G O. Figure 4 shows a photograph of samples consisting of 40 wt, 20 wt and 40 wt of water or glycerol, surfactant HLP, and vegetable oil, respectively. The photograph i in Fig. 4 represents water/ surfactant HLP/vegetable oil system and ii in Fig. 4 represents glycerol/surfactant HLP/vegetable oil system. In the system with water, Wm O was formed Fig. 4-i, whereas in the system with glycerol, Lc L 3 O was formed Fig. 4-ii. Lc L 3 could be seen in the glycerol/surfactant HLP/ vegetable oil system at the low surfactant content region, but was not observed in this region of the water/surfactant HLP/vegetable oil system. Thus, Lc L 3 expanded toward lower surfactant concentration when water was replaced with glycerol in the water/surfactant HLP/vegetable oil system. The expansion of the sponge phase has been observed in previous studies on the ternary system composed of poly- 408

5 Formation of O/W Nano-Emulsion Using Low-Energy Method with Glycerol Fig. 4 Photographs of aqueous solution/surfactant HLP/vegetable oil systems under normal light. Aqueous solutions were composed of water (i) and glycerol (ii). Weight ratio of aqueous solution, surfactant HLP, and vegetable oil was 4:2:4 that is a composition (b) indicated in Fig. 1B. oxyethylene sorbitan monooleate MOPS, 40 w/w sugar solution sucrose, D-fructose, D-glucose, or D-maltose, and vegetable oil 17, 18. Sugars such as D-glucose, D-maltose, and sucrose have been shown to decrease the cloud point of MOPS 27. A decrease in the cloud point of MOPS by adding sugar therefore suggests that the effective HLB of MOPS shifts toward the hydrophobic side in the presence of sugar. Thus, the expansion of the sponge phase region observed in previous studies has been considered to be caused by increased hydrophobicity of MOPS in the presence of sugar. Glycerol also has been shown to decrease the cloud point of octaethylene glycerol dodecyl ether 23. Therefore, we presume that the expansion of the sponge phase or liquid crystal phase region observed in this study is caused by increased hydrophobicity of PGPR and HGML mixtures in the presence of glycerol. Determining the cloud point of PGPR and HGML mixtures was attempted but the cloud point could not be found for either the water or glycerol systems. 3.2 Preparation of nano-emulsion An o/w emulsion was prepared as described in the experimental section Figure 5 shows the positions of the mixtures selected to start emulsification in the phase diagram. By overlaying Fig. 5 onto the phase diagrams Fig. 1A, B, the phase type of the pre-emulsified mixtures was confirmed. Table 1 shows the phase type and weight composition of pre-emulsified samples in the water or glycerol/surfactant Fig. 5 The positions of the mixtures selected to start emulsification in the phase diagram. Compositions, and indicate S/O weight ratios of 8:2, 6:4 and 4:6, respectively. S/O means weight ration of surfactant to oil. HLP/vegetable oil system, and shows the average droplet diameter and PDI of the o/w emulsions prepared from the samples. Figure 6 is a representative of particle size distribution of o/w emulsions prepared from the samples with different S/O weight ratio in the water/surfactant HLP/vegetable oil system A and in the glycerol/surfactant HLP/vegetable oil system B. Figure 7 shows a photograph of the o/w emulsions prepared from samples I to VIII indicated in Table 1. In the case of S/O weight ratio 8:2 in the system with water, when the phase type of pre-emulsified mixtures was Lc L 3 or L 3 La, the average droplet diameter of the prepared o/w emulsions was less than 35 nm and PDI ranged from 0.23 to 0.30 Table 1. In the case of S/O weight ratios 6:4 and 4:6, o/w nano-emulsions with droplet sizes ranging from 40 to 170 nm could be prepared by addition of water to Lc L 3 and PDI ranged from 0.15 to When the phase type of pre-emulsified mixtures was L 3 Wm O, the droplet diameter of the prepared o/w emulsions was more than about 525 nm and PDI ranged from 0.37 to 1.2, and in the case of S/O weight ratio 4:6, the droplet sizes could not be measured. In the case of S/O weight ratio 8:2 in the system with glycerol, when the phase type of pre-emulsified mixtures was Lc L 3, the average droplet diameter of the prepared o/w emulsions was less than 30 nm and PDI ranged from 0.27 to 0.32, and the droplet diameter of the prepared o/w emulsions by addition of water to Lc L 3 G was less than 35 nm and PDI ranged from 0.28 to 0.30 Table 1. In the case of S/O weight ratios 6:4 and 4:6, o/w nano-emulsions 409

6 S. Wakisaka T. Nishimura and S. Gohtani Table 1 Phase type and weight composition of the pre-emulsified samples in the water or glycerol/surfactant HLP/ vegetable oil system and the average droplet diameter and polydispersity index of the prepared o/w emulsions. S/O weight ratio HLP Weight ratio Water for aqueous solution Glycerol for aqueous solution Oil Aqueous solution Phase type Average droplet diameter (nm) Polydispersity index Phase type Average droplet diameter (nm) Polydispersity index Lc+L Lc+L I Lc+L Lc+L II Lc+L Lc+L III 8 : Lc+L Lc+L IV L 3 +La Lc+L V Lc+L Lc+L VI L 3 +Wm+O Lc+L 3 +G VII Wm+O UM* - Lc+L 3 +G VIII Lc+L Lc+L I Lc+L Lc+L II Lc+L Lc+L III 6 : Lc+L Lc+L IV L 3 +Wm+O Lc+L 3 +G V Wm+O Lc+L 3 +G VI Wm+O UM* - Lc+L 3 +G VII Wm+O UM* - Lc+L 3 +G VIII Lc+L Lc+L I Lc+L Lc+L II Lc+L Lc+L 3 +O UM* - III 4 : Wm+O UM* - Lc+L 3 +O UM* - IV Wm+O UM* - Lc+L 3 +O UM* - V Wm+O UM* - Lc+L 3 +G+O VI Wm+O UM* - Lc+L 3 +G VII Wm+O UM* - Lc+L 3 +G VIII UM:unmeasurable level Sample number with droplet sizes ranging from 45 to 115 nm could be prepared by addition of water to Lc L 3 and PDI ranged from 0.17 to When the phase type of pre-emulsified mixtures was Lc L 3 G or Lc L 3 G O, o/w nano-emulsion or o/w emulsions with droplet sizes ranging from 125 to 545 nm could be prepared and PDI ranged from 0.16 to 0.36, and in the case of the preparing o/w emulsions by addition of water to Lc L 3 O, the droplet sizes could not be measured. In both systems, water and glycerol systems, fine o/w nano-emulsions could be prepared by addition of water to Lc L 3, and when oil phase was separated in pre-emulsified samples, fine emulsions could not be prepared. In the case of preparing o/w emulsions by addition of water to Lc L 3 G in the system with glycerol, it was also found that the droplet sizes of emulsions could be relatively small. Replacing water with glycerol was responsible for the expansion of the region containing Lc L 3 toward lower surfactant concentration in the glycerol/surfactant HLP/vegetable oil system; therefore, in the glycerol/surfactant HLP/vegetable oil system compared with the water/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/ w emulsions could be prepared using an isothermal lowenergy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller. Gohtani et al. 11 concluded that identifying and clarifying the phase structure existing in the pre-emulsification and determining the relevant phase structure for producing the desired emulsion size during the emulsification process is very important when employing low-energy emulsification methods. In this study, the role of Lc L 3 in forming o/w nano-emulsions prepared by using an isothermal low-energy method with PGPR and PGFA has been fully described. As described above, PDI of o/w nano-emulsions prepared in this study was more than 0.1, indicating a relatively wide particle size distribution Table 1, and in most cases, particle size distributions showed wide monomodal distribution in S/O weight ratio 8:2 and monomodal or bimodal distribution in S/O weight ratio 6:4 and 4:6 Fig. 6A, B. The appearance of the emulsions was correlated to their mean 410

7 Formation of O/W Nano-Emulsion Using Low-Energy Method with Glycerol Fig. 6 Particle size distribution of o/w emulsions prepared from the samples with different S/O weight ratio in aqueous solution/surfactant HLP/ vegetable oil system. Aqueous solutions were composed of water (A) and glycerol (B). The selected samples were the number (II) for each S/O weight ratio noted in Table 1. particle diameters rather than particle size distributions. The o/w nano-emulsions prepared in S/O weight ratio 8:2 and 6:4 were transparent Fig. 7A I VI, B I VIII and bluish translucent Fig. 7C I IV, D I IV, respectively, in spite of their wide particle size distribution. In S/O weight ratio 4:6, the appearance of formed o/w nano-emulsions or emulsions were milky white Fig. 7E, F ; nevertheless their PDI was relatively low and particle size distributions were slightly narrower than those for the o/w emulsions prepared in S/O weight ratio 8:2 and 6:4 Table 1, Fig. 6A, B. 4 CONCLUSION We have investigated how phase behavior changes by replacing water with glycerol in water/mixture of PGPR and HGML surfactant HLP /vegetable oil systems, and determined the effect of incorporating glycerol in mixtures of PGPR and HGML on o/w nano-emulsion formation using an isothermal low-energy method. In the phase behavior study, a two-phase region with coexisting liquid crystalline Lc and sponge phase L 3 expanded toward lower surfactant concentration when water was replaced with glycerol in a water/surfactant HLP/vegetable oil system. The droplet sizes of the emulsions prepared from a mixture of PGPR and HGML with water or glycerol was examined and it was found that o/w nano-emulsions were formed by emulsification of samples in a region containing Lc L 3. These results indicate that Lc L 3 is an important factor in preparation of o/w nano-emulsions using an isothermal low-energy method with PGPR and PGFA. The PDI of o/w nano-emulsion prepared in this study indicated a relatively wide particle size distribution more than 0.1, however, the appearance of the emulsions could be correlated with their mean particle diameters rather than with their particle size distribution. When weight ratio of surfactant to oil was higher, prepared o/w nano-emulsions had a relatively wide particle size distribution, but their average droplet sizes and appearance were smaller and transparent or bluish translucent, respectively. In the case of lower weight ratio of surfactant to oil, the appearance of prepared o/w nanoemulsions or emulsions were milky white, however, their PDI was slightly less and particle size distribution were slightly narrower. Additionally, in the glycerol/surfactant HLP/vegetable oil system, replacing water with glycerol was responsible for the expansion of the region containing Lc L 3 toward lower surfactant concentration, and as a result, in the glycerol/surfactant HLP/vegetable oil system compared with the water/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/w emulsions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller. Therefore, glycerol was confirmed to facilitate the preparation of nano-emulsions from a system containing surfactant HLP. Moreover, in this study, we could prepare o/w nano-emulsions with a simple one-step addition of water at room temperature without using a stirrer. Thus, the present technique is highly valuable for applications in several industries. References 1 Solans, C.; Izquierdo, P.; Nolla J.; Azemar, N.; Garcia- Celma, M. J. Nano-emulsions. Curr. Opin. Colloid Interface Sci. 10, Chang, Y.; McClements, D. J. Optimization of orange oil nanoemulsion formation by isothermal low-energy methods: influence of the oil phase, surfactant, and temperature. J. Agric. Food. Chem. 62, Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. For- 411

8 S. Wakisaka T. Nishimura and S. Gohtani Fig. 7 Photographs of o/w emulsions prepared using the method described in (A), (C) and (E) indicate the o/w emulsions prepared from S/O weight ratio of 8:2, 6:4 and 4:6, respectively, in the system with water. (B), (D) and (F) shows the o/w emulsions prepared from S/O weight ratio of 8:2, 6:4 and 4:6, respectively, in the system with glycerol. The sample number (I VIII) refers to the number noted in Table

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