A TYROSINASE SUICIDE SUBSTRATE: 8-HYDROXYNARINGENIN 207 semi-preparative C18 reversed-phase column (Spherisorb, 5 μm, 10 i.d. × 250 mm, ODS 2, phase separation). The preparative HPLC conditions were the same as those for analytic HPLC, with the exception of the fl ow rate (3 ml/min) and the injection volume (0.2 ml). The elutions of the metabolite peak were collected and dried under a vacuum. The fi nal purifi ed metabolite (14.2 mg) was analyzed by mass and NMR spectroscopy. INSTRUMENTAL ANALYSIS OF ISOLATED METABOLITES 1 H-NMR spectra were recorded with a Varian Gemini NMR spectrometer at 400 MHz, and 13 C-NMR spectra with a Varian Gemini NMR spectrometer at 100 MHz in DMSO. FAB MS were obtained with a JEOL TMSD-100. The metabolite’s 1 H- and 13 C-NMR spectral data were consistent with those of 8-hydroxynaringenin shown in the literature (14). IRREVERSIBLE INHIBITORY ACTIVITY ASSAY In irreversible inhibitory activity assays, 20 units of tyrosinase was preincubated with 100 μM of the tested samples (dissolved in DMSO) in 1 ml of 50 mM phosphate buffer (pH 6.8) at 25°C. At intervals of 0, 0.5, 2.5, and 5 min, 200 μl of the preincubation mixture was mixed with 800 μl of 2.5 mM L-DOPA. The reaction mixture’s absorbance at 475 nm was monitored every second with a spectrophotometer. The initial reaction rate was measured from the slope of the linear time-dependent increase in absorbance at 475 nm due to the formation of dopachrome produced from L-DOPA by mushroom ty- rosinase. The sample’s relative activity was calculated by dividing its initial rate by that of the control reaction at the beginning, in which DMSO replaced the added sample. DETERMINATION OF THE PARTITION RATIO OF SUICIDE SUBSTRATES OF MUSHROOM TYROSINASE The suicide substrate’s partition ratio was determined according to Waley’s method by incubating 200 μl of preincubation mixture containing 20 units of tyrosinase (0.03 μM) and 0.9–9.0 μM of 8-hydroxynaringenin at 25°C for 30 min (15). Then 800 μl of 2.5 mM L-DOPA was added to each preincubation mixture, and the reaction mixture’s initial rate was determined as described above. Each reaction’s relative activity was calculated by dividing the reaction’s initial rate with the suicide substrate by that of the reaction with- out the suicide substrate. The suicide substrate’s partition ratio could be determined by plotting the fractional activity remaining against the ratio of the initial concentration of the suicide substrate to that of the enzyme. All enzymatic experiments were repeated at least twice in order to ensure the reproducibility of the results, and the mean values ± SD are reported here. RESULTS AND DISCUSSION In an early step of this study, we selected four fl avonoids that belong to different classes including apigenin (fl avone), quercetin (fl avonol), naringein (fl avanone), and catechin as the precursors to study the biotransformation of fl avonoids by A. oryzae. The tested fl avo- noids were added to the medium and cultivated with the fungus. For different cultivation

JOURNAL OF COSMETIC SCIENCE 208 periods, the fermentation broth was extracted with methanol and analyzed via HPLC to identify the presence of any metabolite. Among the tested fl avonoids, we found that nar- ingenin could be biotransformed by A. oryzae during the fermentation process (data not shown). We then scaled up the fermentation quantity and purifi ed the metabolite by ethyl acetate extraction, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC methods. The NMR and mass spectral analysis confi rmed the metabolite as 8-hydroxynaringenin (Figure 1) in comparing the spectral data with those of previ- ous reports (14). Our previous work demonstrated two 8-hydroxyl fl avonoid derivatives, 8-hydroxydaidzein and 8-hydroxygenistein, to be potent suicide substrates of mushroom tyrosinase (16). Thus, it is interesting to test the suicide substrate properties of 8-hydroxynaringenin toward mushroom tyrosinase. Because suicide substrates belong to irreversible inhibitors, we then determined the irreversible inhibitory activity of 8-hydroxynaringenin toward mushroom tyrosinase by preincubation experiments. The result is shown in Figure 2. The enzymatic activity remained constant during fi ve minutes in the preincubation mix- tures without the tested samples (control) or with 100 μM of naringenin. However, preincubation of tyrosinase with 100 μM of 8-hydroxynaringenin quickly inactivated the enzyme within the fi rst 30 seconds of preincubation and only 1.5% residual activity remained in the preincubation mixture after fi ve minutes of preincubation. These results Figure 1. Chemical structures of naringenin and 8-hydroxynaringenin. Figure 2. Relative activities of preincubations of mushroom tyrosinase with naringenin (□), 8-hydroxynar- ingenin (▲), and without a sample (●) for varying durations of preincubation time.