REDUCTION OF HYPERPIGMENTATION BY A. INCISUS EXTRACT 5 was added to the fi nely dispersed emulsion. Finally, TEA was added to neutralize the carbopol polymer. The effect of the contents of the emulsifi er and co-emulsifi er on the droplet size was also investigated in this study. The contents of the emulsifi er (4–16% w/w) and co-emulsifi er (1–8% w/w) were varied, and the formula that provided the smallest droplet size would be selected for stability studies and in vivo studies of UVB-induced hyperpigmentation in C57BL/6 mice. CHARACTERIZATION OF THE FORMULATED NANOEMULSIONS Morphology. The morphology of the nanoemulsions was observed using transmission elec- tron microscopy (TEM, Technai F20, Philips, Eindhoven, The Netherlands). To perform the TEM observations, the nanoemulsions were diluted with deionized water. A drop of the diluted nanoemulsions was then applied to carbon-coated grids. Two minutes later, the excess was drawn off with fi lter paper. A saturated uranyl acetate aqueous solution was used as a staining agent. After air drying, the morphology of the sample was observed by TEM at a magnifi cation of 370 kx. Droplet size analyses. The mean droplet size and polydispersity value of the nanoemulsions were analyzed by photon correlation spectroscopy (PCS), employing a Zetasizer (Model Nano ZS90, Malvern instruments Ltd., Malvern, Worcestershire, UK). An aliquot of the nanoemulsions was re-suspended in water. Measurements were performed at a fi xed angle of 90° to the incident light, and data were collected over a period of 3 min. Three mea- surements were performed separately on each prepared sample. Viscosity determination. The viscosity of the nanoemulsions was determined by a viscometer (model DV-III, Brookfi eld, Arizona) equipped with a cone and plate (plate diameter 40 mm, cone angle 4°). Three measurements were carried out at a temperature of 30°C with a torque of 10 rpm and were performed separately on each prepared sample. pH measurement. The pH of the nanoemulsions was measured using a pH meter (Model Delta 320, Mettler Toledo, Guangzhou, China). Three measurements were made sepa- rately on each prepared sample. STABILITY OF THE SELECTED NANOEMULSION FORMULA CONTAINING A. INCISUS EXTRACT The stability of the nanoemulsions containing A. incisus extract was studied under normal and accelerated conditions. For the normal condition, the selected formula was stored at room temperature (35° ± 3°C, during summer at Phitsanulok Province, Thailand) for three months. For the accelerated condition, the nanoemulsion was subjected to seven heat-cool cycles (4°C for 24 h and alternated to 45°C for another 24 h). The quantity of artocarpin was determined by the HPLC method described above. Moreover, the physical appearances of the nanoemulsions were examined in terms of viscosity, droplet size, and pH value. IN VIVO STUDY ON THE DEPIGMENTING EFFICACY OF THE SELECTED NANOEMULSION FORMULA IN UVB-INDUCED HYPERPIGMENTATION OF C57BL/6 MICE Three male C57BL/6 mice (National Laboratory Animal Centre, Mahidol University, Bangkok, Thailand), aged fi ve weeks and weighing 20–25 g, were used. The mice were
JOURNAL OF COSMETIC SCIENCE 6 housed on sawdust, three per cage, with free access to food and tap water. Room illumi- nation was on an automated cycle of 12 h darkness, and room temperature was main- tained at 25° ± 2°C. They were acclimatized to the laboratory conditions for at least one week prior to the experiments. UVB-induced hyperpigmentation was induced on the backs of the C57BL/6 mice by fol- lowing previous studies (15,16) with modifi cation. The mice were anesthetized with 5.47 g/ml solution of pentobarbital sodium (0.8 ml/kg). The back hair of the animal was shaved with a razor in the tail-to-head direction without damaging the skin. A bank of fi ve fl uorescent sunlamps (Toshiba FL8BLB, Japan) emitting rays between 275 and 305 nm, with a peak at 305 nm, was used as a UVB source. The lamp-to-skin distance was 30 cm. Two separate areas (2 × 2 cm) on the back of each animal were irradiated with 91.4 J/cm2 per exposure from the UVB lamps for ten days. The formulated nanoemulsion was topi- cally applied to one of the irradiated areas twice a day for six weeks, while the aqueous solution of the extract (0.02% w/w) was applied to the other one. The skin color of the treated area was evaluated by a Mexameter MX® 18 (Courage and Khazaka Electronic GmbH, Cologne, Germany). Once every week from the beginning of sample applications, the degree of depigmentation was evaluated as the melanin value (M), estimated as follows: ' M=M (at day 0) M(at each day measured) The M value expresses the melanin value or degree of hyperpigmentation and the high value of ΔM indicates the depigmenting effi cacy of the formula. STATISTICAL ANALYSIS A Student’s t-test was used for comparison between the averages of two independent groups. A p-value of equal to or less than 0.05 was considered statistically signifi cant. RESULTS AND DISCUSSION QUANTITY OF ARTOCARPIN IN THE EXTRACT The appearance of A. incisus extract was a yellow powder. According to the HPLC method, the amount of artocarpin in the extract was determined to be 44.5 ± 0.1% w/w. The obtained result corresponded with that of our previous study, indicating 45.2 ± 0.5% w/w of artocarpin in the extract (5). IC50 VALUE OF MELANOGENESIS-INHIBITORY ACTIVITY OF THE EXTRACT IN MOUSE MELANOCYTE CELLS The A. incisus extract exhibited a dose-dependent inhibition of melanin production in B16F1 cells, as shown in Figure 1. At concentrations of 10, 15, 25, 40, 80, and 100 μg/ ml of A. incisus extract, the percentage of melanin reduction compared to the control was
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