J. Cosmet. Sci., 62, 1–14 ( January/February 2011) 1 Depigmenting action of a nanoemulsion containing heartwood extract of Artocarpus incisus on UVB-induced hyperpigmentation in C57BL/6 mice SUPASIRI BURANAJAREE, PIYAPORN DONSING, RATTIMA JEENAPONGSA, and JARUPA VIYOCH, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok, 65000 Thailand (S.B., J.V.) Borntras (Thailand) Ltd., 142-143 Moo 6, Chiangraknoi, Bang Pa-in District, Phra Nakhon Si Ayutthaya, 13180 Thailand (P.D.) Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok, 65000 Thailand (R.J.) and Cosmetics and Natural Products Research Center (CosNat), Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok, 65000 Thailand ( J.V.). Accepted for publication September 30, 2010. Synopsis Melasma hyperpigmentation is an acquired disorder predominantly affecting the female population. The present study was conducted to determine the potential of a botanical extract to reduce observable hyperpig- mentation. The extract from heartwood of Artocarpus incisus was formulated into nanoemulsions, and the de- pigmenting effi cacy of the formulated nanoemulsion was determined in vivo. HPLC analysis showed that the extract contained artocarpin in an amount of 44.5 ± 0.1% w/w. The extract exhibited melanogenesis inhibi- tion with an IC50 value of 30.2 ± 2.4 mg/ml, while kojic acid, a well known lightening agent, exhibited an IC50 of 51.4 ± 5.1 mg/ml. The nanoemulsion containing the extract was then formulated and prepared by the phase inversion technique. The concentration of the extract used was about six times its IC50. The optimal formula containing 0.02% w/w extract, 41.6% w/w isopropyl myristate, 0.03% w/w α-tocopherol, 5% w/w glyc- eryl monostearate (co-emulsifi er), 8% w/w ceteareth-10 (emulsifi er), 0.05% triethanolamine, 0.03% w/w carbopol 940, and water adjusted to 100% w/w provided a homogeneous o/w emulsion with a droplet size of 325 ± 15 nm and a polydispersity of 0.31 ± 0.02. The depigmenting effi cacy was then observed following topical application of the formulated nanoemulsion to UVB-stimulated hyperpigmented dorsal skin of C57BL/6 mice. A strongly visible decrease in hyperpigmentation was observed after six weeks of treatment with the formulated nanoemulsion. The degree of pigmentation decreased after the application was 84 ± 4 units, while that after the application of the extracted prepared into solution was 51 ± 3 units. The applied areas would return to their original color after treatment was stopped for four weeks. Address all correspondence to Jarupa Viyoch at jarupav@nu.ac.th or at jarupaviyoch4@yahoo.com.
JOURNAL OF COSMETIC SCIENCE 2 INTRODUCTION Melasma is a common acquired hyperpigmentary disorder characterized by dark patches or macules located on the cheeks, forehead, upper lip, chin, and neck. Pigmentation is pre- dominantly found in females, accounting for 90% of all cases (1,2). It appears in all racial types, but it occurs more frequently in persons with darker complexions (Fitzpatrick’s skin type IV) (3,4), especially those living in high-ultraviolet-radiation areas, including Thailand. Although melasma is a disfi guring skin condition that worsens with sunlight, it is considered to be a cosmetic problem, as there is no pain or other associated symptoms. Nowadays, safe and effective components extracted from natural sources have been re- ported for their potential application in improving hyperpigmentation disorders. These extracts are mostly free from harmful side effects. For this reason, there is an increasing interest in fi nding natural extracts for application in personal care products and cosmetics. Our previous study revealed that the extract from heartwood of Artocarpus incisus (bread- fruit or Sa-kae in Thai) provided tyrosinase inhibitory and anti-oxidation activities (5). Additionally, it was found that artocarpin, the major component of A. incisus extract, exhibited skin lightening effects on the UVB-induced hyperpigmented dorsal skin of brown- ish guinea pigs (6,7). These fi ndings indicated the extract from A. incisus heartwood to be a potential source of depigmenting agents and that its site of action is in the substratum, in particular within the epidermis and hair follicles. Therefore, to increase the effi cacy of the extract, the tools for increasing penetration of the extract through skin are needed. As we know, delivery systems play an important role in the fi elds of cosmetics and dermophar- maceuticals. The key aspects of the delivery systems are increasing and/or improving penetra- tion and effi cacy, controlling delivery, separating incompatible actives, prolonging shelf life, and/or decreasing the degradation of active compounds. Nanoemulsions have increasingly become one of the most popular delivery systems. Nanoemulsions can be defi ned as oil- in-water (o/w) emulsions, with mean droplet diameters ranging from 50 to 1,000 nm (8). They can be “transparent” (mean droplet size 200 nm) or “milky” (mean droplet size 500 nm) (9,10). Nanoemulsions would infl uence the transport properties of the active ingredients since their compositions such as emulsifi ers and co-emulsifi ers can enhance the skin penetra- tion of active ingredients and increase their concentrations in the skin. Additionally, due to smaller particle sizes, they offer higher stability against creaming or sedimentation as com- pared to macroemulsions (10). Furthermore, nanoemulsions are more suitable for the trans- portation of lipophilic compounds, according to the lipophilic interior of nanoemulsions. Therefore, in the present study, the natural extract from heartwood of A. incisus was for- mulated into various nanoemulsion formulations. An in vivo study of UVB-induced hyper- pigmentation in C57BL/6 mice was performed to investigate visible results of the formulated nanoemulsion in application for the purpose of depigmenting. The results from this study showed that a nanoemulsion containing heartwood extract from A. incisus could remarkably improve the hyperpigmented lesion on the dorsal skin of C57BL/6 mice. MATERIALS AND METHODS PREPARATION OF HEARTWOOD EXTRACT OF A. INCISUS The heartwood of A. incisus was collected in July 2007 from Phitsanulok Province, Thai- land. To prepare the extract, the heartwood portion was chipped, exposed to the sun, and
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