REDUCTION OF HYPERPIGMENTATION BY A. INCISUS EXTRACT 11 DEPIGMENTING EFFICACY OF THE SELECTED NANOEMULSION FORMULA IN UVB-INDUCED HYPERPIGMENTATION OF C57BL/6 MICE Figure 7 shows photographs of the depigmenting effect on the dorsal skin of the C57BL/6 mouse. As shown in Figure 7C, after treatment with the nanoemulsion containing the extract for four weeks, a greater decrease in hyperpigmentation was observed when com- pared with the extract prepared as a solution. Furthermore, a strongly visible decrease in hyperpigmentation was observed at week 6, as shown in Figure 7D. The melanin values measured by a Mexameter MX® 18 at the UV-induced hyperpigmented dorsal skin areas from the day after tanning through the six weeks of application are shown in Figure 8A. The nanoemulsion containing the extract decreased the degree of pigmentation (ΔM-value) by 84 ± 4 units while the extract solution decreased by 51 ± 3 units (Figure 8B). This indicated that the extract formulated into a nanoemulsion provided a higher skin- depigmenting potential than that prepared in solution form. Higher effi cacy of the nano- emulsion is probably due to destabilization of the bilayer structure of the stratum corneum by permeation enhancers like IPM, ceteareth-10, and the GMS contained in the formula. Figure 7. Effect of the selected nanoemulsion formula containing 0.02% w/w extract or the solution con- taining 0.02% w/w extract on improvement of UVB-induced hyperpigmentation in C57BL/6 mouse skin. All photographs were from the same animal: (A) before UV radiation (B) initial pigmentation after UV ir- radiation (C,D) after topical application for four weeks and six weeks, respectively and (E) after application had been stopped for four weeks.

JOURNAL OF COSMETIC SCIENCE 12 Generally, most of the lipophilic agents pass the stratum corneum through an intercellular lipid bilayer pathway (24). The destabilization of such a lipid bilayer will enhance the lipid pathway’s permeability to lipophilic agents such as artocarpin, the major component of the extract. Moreover, the small droplet size and low viscosity of the nanoemulsion make it an excellent carrier for enhancing percutaneous uptake of lipophilic compounds through the stratum corneum and/or hair follicles. This is because the number of internal droplets that can interact on a fi xed area of the targeted sites will increase when the drop- let size and viscosity decrease (25). Therefore, the higher effi cacy of the nanoemulsion formulation could be the result of the combined effects of the enhancers of skin perme- ation as well as the small droplet size of the nanoemulsions. As mentioned previously, artocarpin has depigmenting effects on UVB-induced hyper- pigmentation on the dorsal skin of brownish guinea pigs (7). In the present study, there- fore, it is possible to conclude that the melanogenesis-inhibitory activity of A. incisus extract is also involved with artocarpin. Generally, the ideal of depigmenting agents should have a potent, rapid, and selective bleaching effect on hyperactivated melanocyte cells and carry no short- or long-term side effects. The present in vivo study showed that reduced hyperpigmentation was observed following topical applications of the extract for four weeks and that the treated areas (UVB-induced hypigmentation) could return to close to their original color after stopping treatment for four weeks (Figure 7E). This fi nding implies that no permanent dysfunction of skin cells is caused by the extract of A. incisus when the optimal concentration is used. Furthermore, visible edema or scaling was Figure 8. (A) Averaged melanin values measured by a Mexameter MX® 18 at the UV-induced hyperpig- mented dorsal skin areas from the day after tanning (week 0) through six weeks of application. (B) The degree of depigmentation (ΔM-value) after application of the prepared nanoemulsion containing 0.02% w/w extract or the solution containing 0.02% w/w extract for six weeks. Each point or bar represents mean ± S.D. (n = 3). Student’s t-test showed a signifi cant difference between the two formulations, ** p0.01.