J. Cosmet. Sci.) 59, 41-58 CTanuary/February 2008) Evaluation of the effect of Thai breadfruit's heartwood extract on melanogenesis-inhibitory and antioxidation activities PIYAPORN DONSING, NANTEETIP LIMPEANCHOB, and JARUPA VIYOCH, Department of Pharmaceutical Technology) Faculty of Pharmaceutical Sciences (P.D.J J. V.)! Department of Pharmacy Practice! Faculty of Pharmaceutical Sciences (N.L.)! and Cosmetic and Natural Product Research Center! Naresuan University Hospital U. V.)! Naresuan University! Phitsanulok! 65000 Thailand. Accepted for publication September 14! 2007. Synopsis The aim of this study was to clarify the melanogenesis-inhibitory and antioxidant activity of Thai bread­ fruit's heartwood extract for application as a skin-lightening agent. The heartwood of breadfruit (Artocarpus incisus) grown in Phitsanulok Province, Thailand, was extracted by using diethyl ether or methanol. The amount of artocarpin, a major component of A. incisus extract, was determined by using the HPLC method. The artocarpin content found in ether extract was 45.19 ± 0.45% w/w, whereas that in methanol extract was 19.61 ± 0.05% w/w. The ether extract was then evaluated for tyrosinase-inhibitory, melanogenesis­ inhibitory, and antioxidant activities. The tyrosinase-inhibitory activity was tested in vitro by monitoring the inhibition of the extract against the formation of DOPAchrome by tyrosinase enzyme. The results showed that the tyrosinase-inhibitory activity of the extract was in a dose-dependent manner. The obtained IC50 value was 10.26 ± 3.04 µg/ml, while kojic acid, a well-known tyrosinase inhibitor, provided an IC50 of 7.89 ± 0.18 µg/ml. Melanocyte B16Fl melanoma cells (ATCC No. CRL-6323) were then used for determination of the melanogenesis-inhibitory activity of the extract, comparing it to hydroquinone, kojic acid, and purified artocarpin. The amount of melanin produced by the cells was monitored by measuring an absorbence at 490 nm. The obtained results indicated that A. incisus extract at a concentration of 2 to 25 µg/ml was able to decrease the melanin production of the melanocyte B16Fl cells. The obtained micrograph also confirmed that the extract did not change the cell morphology but reduced the melanin content by inhibiting melanin synthesis, whereas the purified artocarpin at a concentration of 4.5 µg/ml caused changes in cell morphology. Additionally, the extract exhibited antioxidant activity in a dose­ dependent manner at an EC 50 of 169.53 ± 9.73 µg/ml, according to DPPH assay. The obtained results indicated that the ether extract of A. incisus's heartwood has the potential of acting as a skin-lightening agent for application in cosmetics. INTRODUCTION In the cosmetic industry, the demand for multifunctional products and their efficiency are the keys to trends in technology, innovations, and the cosmetic market. Skin- Address all correspondence to Jarupa Viyoch. 41

42 JOURNAL OF COSMETIC SCIENCE lightening products are widely used for cosmetic purposes. The ideal lightening agents should have a potent, rapid, and selective bleaching effect on hyperactivated melanocyte cells and carry no short- or long-term side effects. Interference with the melanin synthesis of the lightening agents can be achieved by regulating the activity of melanogenic enzymes, the distribution of melanosomes, and the turnover of pigmented keratinocytes. Most lightening agents act specially to reduce the function of tyrosinase, which is the key enzyme in melanin biosynthesis (1). Nowa­ days, several researchers have reported that the lightening effects relate to antioxidant properties. Compounds with redox properties can have depigmenting effects by inter­ acting with o-quinones and avoiding the oxidative polymerization of melanin interme­ diates. The phenolic derivatives of flavonoids are the most important antioxidants that can chelate the copper ions in tyrosinase. These indicate that the antioxidants can inhibit the activity of the tyrosinase enzyme and could be effective as lightening agents (1). Recently, safe and effective tyrosinase inhibitors extracted from natural sources have been reported for their potential applications in improving hyperpigmented disorders. For example, extracts from Glycyrrihiza glabra (licorice), Morus alba L. (white mulberry), Carthanus tinctorius L. (safflower), Arctostaphylos Uva-Ursi (bearberry), and Oryza Sativa (rice bran) have been used as skin-lightening agents. These materials are mostly free from harmful side effects. For this reason, there is an increasing interest in finding natural tyrosinase inhibitors from natural sources. Artocarpus incisus (breadfruit) belongs to the Moraceae family. This evergreen tree, called "Sa-Ke" in Thai, is found throughout the tropical world. Because its pulp contains a high content of carbohydrate in the amount of 76.7%, it has been used as an important source of energy over the years (2). It has been reported that the methanol extract of the heartwood of A. incisus grown in Okinawa, Japan, strongly inhibits tyrosinase activity (3). Additionally, the mother liquor from crystallization of A. incisus ether extract also shows a melanin biosynthesis-inhibitory effect on brown guinea pigs. The heartwood extract of A. incisus consists of several flavonoids including artocarpin, ( + )-norartocarpin, artocarpesin, ( + )-dihydromorin, chlorophorin, ( + )-norartocapanone, artocarbene, 4-prenyloxyresveratrol, and cycloartocarpin. Among these compounds, chlorophorin, ( + )-norartocapanone, artocarbene and 4-prenyloxyresveratrol show much higher tyrosi­ nase-inhibitory activity than kojic acid, whereas artocarpin does not show tyrosinase­ inhibitory activity. However, this compound shows a skin-lightening effect on the DVB-induced hyperpigrnented dorsal skin of brownish guinea pigs (4). It is likely that the A. incisus extract inhibits the melanogenesis process of melanocytes through other pathways. These findings lead to the question: does the crude extract exhibit higher rnelanogenesis-inhibitory activity than the purified artocarpin? Since the extract con­ tains several kinds of flavonoids, the synergistic effects of tyrosinase-inhibitory, antioxi­ dant, and other possible activities would be taking place. Therefore, the aim of this research was to evaluate the potential of A. incisus crude extract for application as a skin-lightening agent. The heartwood of A. incisus was extracted by using various organic solvents, and the obtained extracts were then used to determine the amount of artocarpin, a major component of A. incisus extract, by using high­ performance liquid chromatography (HPLC). The inhibitory effect of the extracts on melanin biosynthesis was investigated by using in vitro DOP Achrorne assay and a cell culture model. Additionally, an in vitro DPPH assay was performed to determine the