JOURNAL OF COSMETIC SCIENCE 250 did not obviously decrease the droplet size of emulsion. These results agree with other studies that discuss the relationship between surfactant concentration and droplet size of emulsion (12,15). THE EFFECT OF HLB VALUES The emulsion was prepared by using the mixed surfactant of sorbitane trioleate and poly- oxyethylene (10) oleyl ether. The So ratios were adjusted to satisfy the proper HLB values for optimum emulsifi cation condition. Emulsions with 10.0 wt% D -limonene and So ra- tio at 0.4 were prepared at different HLB values. The relationship between the droplet size of emulsions and the HLB values is shown in Table I. The results showed that droplet size of emulsions can be affected by the HLB values. The droplet size of emulsion was 332 nm at the HLB value of 2 and with an increase in the HLB value to 12, the droplet size decreased to 23 nm. The proper HLB values of mixed surfactants were key factor for the formation of emulsion droplets. During the formation of O/W emulsion, the lipophilic surfactants have more affi nity to dispersed droplet of emulsion than the hydrophilic sur- factant. A proper HLB value is needed to maintain the oil phase and water phase equilib- rium, and with optimum HLB values, it can stabilize and narrow down the newly formed droplets during the emulsifi cation. The appearance of emulsion is milk-like at HLB val- ues of 2–10, and transparent or translucent to the naked eye at HLB value of 12. TEM OBSERVATION To observe the physical properties of the nanostructured droplet in the nanoemulsion system, TEM analysis was carried out with negatively stained samples. The results are shown in Figure 4, where phosphotungstic acid–stained D -limonene droplets are clearly Table I The Effect of HLB Values on the Emulsion Droplet Size and Appearance of D -Limonene in Water Emulsion System No. HLB values Droplet size (nm) Appearance 1 2 332 ± 27.3 Milk-like 2 3 335 ± 22.12 Milk-like 3 4 287 ± 14.21 Milk-like 4 5 226 ± 13.52 Milk-like 5 6 207 ± 7.33 Milk-like 6 7 175 ± 2.58 Milk-like 7 8 149 ± 3.14 Milk-like 8 9 102 ± 2.11 Milk-like 9 10 73 ± 1.29 Milk-like 10 11 54 ± 0.67 Milk-like 11 12 23 ± 0.86 Translucent So ratio = 0.4 applied power = 18 W ultrasonic time = 120 s.

NANOEMULSION OF D-LIMONENE IN WATER SYSTEM 251 visible and the droplet size correlated well with the results from droplet size analysis us- ing Nanotrac 150 light scattering instrument. In addition, the morphology of the D - limonene droplet was spherical, and the gray parts of the droplet were D -limonene precipitation incorporated in emulsion system. CONCLUSION Nanoemulsion droplet can be obtained in D -limonene in water system by ultrasonic emulsifi cation. The optimum conditions for ultrasonic emulsifi cation of D -limonene na- noemulsions were applied power of 18 W, ultrasonic time of 120 s, and So ratio of 0.6 with an HLB value below 12. Nanoemulsion droplet in D -limonene nanoemulsion system was probably a solution for solving the low bioavailability and low solubility problems of D -limonene and thus might be applied as nanoencapsulated fl avor and fragrance systems for industry. ACKNOWLEDGMENT The fi nancial support (NSC 98-2313-B-002-042-MY3) provided from the National Science Council, the Republic of China (Taiwan) is gratefully acknowledged. REFERENCES (1) C. Solans, J. Esquena, A. M. Forgiarini, N. Usón, D. Morales, P. Izquierdo, N. Azemar, and M. J Garcia- Celma, Nanoemulsions: Formation, properties and applications, J. Surf. Sci. Series, 109, 525–554 (2003). (2) L. Weirong, S. Dejun, L. Caifu, L. Qian, and X. Jian, Formation and stability of paraffi n oil-in-water nano- emulsions prepared by the emulsion inversion point method, J. Colloid Interf. Sci., 303, 557–563 (2006). (3) C. M. Pey, A. Maestro, I. Solé, C. González, C. Solans, and J. M. Gutiérrez, Optimization of nano- emulsions prepared by low-energy emulsifi cation methods at constant temperature using a factorial design study, Colloids Surf. A, 288, 144–150 (2006). Figure 4. Visual appearance and transmission electron micrographs of nanostructured droplet in D -limonene in water emulsion system. The scale bar represents a distance of 50 nm.