J. Cosmet. Sci., 61, 205–210 (May/June 2010) 205 Identifying 8-hydroxynaringenin as a suicide substrate of mushroom tyrosinase TE-SHENG CHANG, MENG-YI LIN, and HSUAN-JUNG LIN, Department of Biological Science and Technology, National University of Tainan, 33 sec. 2, Su-Lin St., Tainan, Taiwan. Accepted for publication September 28, 2009. Synopsis A biotransformed metabolite of naringenin was isolated from the fermentation broth of Aspergillus oryzae, fed with naringenin, and identifi ed as 8-hydroxynaringenin based on the mass and 1 H- and 13 C-NMR spectral data. The compound showed characteristics of both an irreversible inhibitor and a substrate of mushroom tyrosinase in preincubation and HPLC analysis. These results demonstrate that 8-hydroxynaringenin belongs to a suicide substrate of mushroom tyrosinase. The partition ratio between the compound’s molecules in the formation of product and in the inactivation of the enzyme was determined to be 283 ± 21. The present study’s results, together with our previous fi ndings, which proved that both 8-hydroxydaidzein and 8-hydroxy- genistein are suicide substrates of mushroom tyrosinase, show that 7,8,4′-trihydroxyl functional groups on fl a- vonoids’ skeletons play important roles in producing suicide substrate properties toward mushroom tyrosinase. INTRODUCTION Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase widely distributed in nature. This enzyme catalyzes the fi rst two reactions of melanin synthesis, the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine, L-DOPA, and the oxidation of L-DOPA to dopaquinone. This o-quinone is a highly reactive compound and can polymerize sponta- neously to form melanin (1). The enzyme also is known as a polyphenol oxidase (PPO) and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing, which are caused by the oxidation of phenolic compounds in the fruits (2). Both the hyperpigmentation in skin and this enzymatic browning in fruits are undesirable. Hence, inhibiting the tyrosinase activity has been the subject of many studies (3). Flavonoids are a diverse group of polyphenolic compounds widely distributed in plants with wide-ranging biological properties. Some of them have been demonstrated to be effective inhibitors of mushroom tyrosinase (4–11). In our continuing search for tyrosi- nase inhibitors from fl avonoids, we isolated seven isofl avones from soygerm koji fermented by Aspergillus oryzae and demonstrated their potent inhibitory effects on mushroom tyrosinase Address all correspondence to Te-Sheng Chang.

JOURNAL OF COSMETIC SCIENCE 206 (12,13). Among them, two hydroxylated soy isofl avone derivatives, 8-hydroxydaidzein and 8-hydroxygenistein, biotransformed by A. oryzae, possessed a vastly higher inhibitory activity than their precursors, the soy isofl avones daidzein and genistein. Based on our previous fi ndings, we are interested to know whether other kinds of fl avonoids could also be biotransformed by A. oryzae. If the answer is yes, what are the metabolites’ effects on mushroom tyrosinase activity? In the present study, a biotransformed metabolite of nar- ingenin was isolated from the fermentation broth of Aspergillus oryzae fed with naringenin and identifi ed as 8-hydroxynaringenin based on the mass and 1 H- and 13 C-NMR spectral data. The inhibitory property of the metabolite toward mushroom tyrosinase was inves- tigated. MATERIALS AND METHODS MICROORGANISMS AND CHEMICALS Lyophilized culture of A. oryzae BCRC 32288 was obtained from the Bioresources Collec- tion and Research Center (BCRC) of the Food Industry Research and Development Insti- tute (FIRDI, Hsinchu, Taiwan, ROC). The stock culture was grown on potato dextrose agar (PDA) and maintained at 25°C. Spore suspension of A. oryzae was prepared in sterile water and used for inoculation. Mushroom tyrosinase (5370 U/mg), L-DOPA, dimethyl sulfoxide (DMSO), Sephadex LH-20 gel, and naringenin were purchased from Sigma Chemical (St. Louis, MO). Yeast extract, malt extract, peptone, agar, and potato dextrose agar (PDA) were obtained from Difco Laboratories (Detroit, MI). High-performance liq- uid chromatography (HPLC)-grade acetonitrile and acetic acid were obtained from J.T. Baker (Phillipsburg, NJ). The other reagents and solvents used are commercially avail- able and were used as received. FUNGAL CULTIVATION AND METABOLITE PURIFICATION The cultivation condition and the medium used for A. oryzae BCRC32288 were exactly according to the data sheet from the BCRC of the FIRDI. The fermentation experiments were carried out in 250-ml baffl ed Erlenmeyer fl asks containing 20 ml of the medium in the presence of 250 μg naringenin per ml of the medium. Two liters of the cultivations were carried out for metabolite purifi cation. Cultures were incubated for two days with a rotary shaker at a speed of 120 rpm/min and 30°C then the cultivations were combined and extracted with two liters of ethyl acetate. The ethyl acetate extract was dried under a vacuum. The dry mass (5.8 g) was resuspended with 10 ml of methanol and then fraction- ated by Sephadex LH-20 gel column chromatography (50 cm × 2.6 i.d.) with methanol as an eluent. Every 50 ml of elution was collected and 20 μl of each fraction was analyzed by HPLC to identify the metabolite’s presence. The operational conditions for the HPLC analysis by an analytic C18 reversed-phase column (Spherisorb, 5 μm, 4.6 i.d. × 250 mm, ODS 2, phase separation, Deeside Industrial Park, Clwyd, UK) consisted of an isocratic elution for 15 min with 35% acetonitrile in 1.0% (v/v) acetic acid at a fl ow rate of 1 ml/ min, and detection of absorbance at 280 nm. Fractions 11 to 15 from Sephadex LH-20 gel column chromatography were identifi ed to contain the metabolite and were dried under a vacuum. The dried mass (2.1 g) was then purifi ed by repeated HPLC using a