JOURNAL OF COSMETIC SCIENCE 348 stagnated blood in female genital diseases (5) and for skin care, as mentioned in ancient books (6). Earlier investigations have reported the pharmacological activity of P. suffruti- cosa and the isolation of acetophenones, monoterpenes, monoterpene glycosides, triterpe- noids, aromatic acid derivatives, fl avonoids, steroids, and purine from the root cortex of this plant (7–8). In previous research we also reported the isolation, structural identifi ca- tion, and pharmacological activity of some natural compounds from P. suffruticosa (9–12). This paper deals with the isolation and identifi cation of tyrosinase inhibitors from the root cortex of P. suffruticosa. MATERIALS AND METHODS GENERAL EXPERIMENTAL PROCEDURES Melting points were determined on a Yanagimoto micro-melting point apparatus and are uncorrected. Optical rotations were determined on a JASCO DIP-370 polarimeter at 25°C. The UV spectra were obtained on a Hitachi 200-20 spectrophotometer, and IR spectra were measured on a Hitachi 260-30 spectrophotometer. 1 H NMR spectra were recorded with a Varian Gemini NMR spectrometer at 400 MHz, and 13 C NMR spectra were recorded with a Varian Gemini NMR spectrometer at 100 MHz in CDCl3, CD3OD, and C5D5N. EIMs were obtained with a JEOL JMS-HX110 mass spectrometer at 70 eV, and FABMs were obtained with a JEOL TMSD-100 mass spectrometer. Silica gel (Merck, 60–230 or 230–400 mesh) and Sephadex LH-20 were purchased from Pharmacia Fine Chemicals. A pre-packed column (Lihroprep RP-8, 40–63 mm, 250 × 310 μm) of low- pressure liquid chromatography was purchased from Merck and Co. Preparative HPLC was carried out by a Shimadzu LC-8A chromatograph on a reverse phenyl column (Shim- pack pre-phenyl column, 20 × 250 mm, 10 μm) for reverse chromatography (10). MATERIALS The root cortex of P. suffruticosa was purchased from a local Chinese drug store (Chen Yen Company, Taipei, Taiwan). The specimen of the plant was verifi ed by Prof. Sheng Chu Kuoh (China Medical University, Taichung, Taiwan) via morphological examinations with a light microscope. Mushroom tyrosinase (2870 U/mg), L-tyrosine, L-Dopa, and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical (St Louis, MO). Kojic acid was obtained from Aldrich Chemical Co. (Milwaukee, WI). Other reagents and sol- vents used were commercially available and used as received. EXTRACTION AND SEPARATION The dry root cortex (45.0 Kg) of P. suffruticosa was crushed and extracted four times with 95% EtOH, followed by 70% EtOH. The combined EtOH extracts (6.8 kg) were concen- trated under reduced pressure to yield dark brown syrup that was partitioned between 90% MeOH (MeOH/H2O = 9:1) and n-hexane. The concentrated 90% MeOH layer (5.09 kg) was concentrated and partitioned with EtOAc and H2O. The aqueous solution was again

TYROSINASE INHIBITORS FROM P. SUFFRUTICOSA 349 partitioned between n-BuOH and H2O. Each solute layer was concentrated in a vacuum to yield n-hexane (1.61 kg), ethyl acetate (0.178 kg), n-butanol (0.89 kg), and water (3.02 kg). The ethyl acetate layer and n-BuOH (100 mg/ml in DMSO) showed the high- est anti-tyrosinase activity. The ethyl acetate layer was subjected to silica gel column chromatography and eluted with CHCl3, CHCl3-MeOH (97:3) to afford acetophenones. The residue was eluted with MeOH, combined with the n-BuOH extract, and subjected to column chromatography on silica gel with CH2Cl2-MeOH (95:5, 90:10, 85:15) and MeOH, successively. Rechromatography of the fi rst fraction on a Sephedex LH-20 col- umn, eluted with MeOH, gave two fractions. The fi rst fraction was further separated by a Sephedex LH-20 column, eluted with MeOH-H2O (95:5), to give kaempferol (com- pound I, 25.0 mg) and quercetin (compound II, 40.5 mg). Rechromatography of the second fraction on preparative Lobar RP-8, followed by HPLC separation using a phenyl column and MeOH-H2O (45:55, 35:65) as eluted, gave mudanpioside B (compound III, 186.3 mg), benzoyloxy-paeonifl orin (compound IV, 5.04 gm), and mudanpioside H (compound V, 366.0 mg). In the same manner, the third fraction using MeOH-H2O (20:80) as eluted gave pentagalloyl-β-D-glucose (compound VI, 67.5 mg). ENZYMATIC ASSAY OF TYROSINASE Mushroom tyrosinase activity was determined according to the methods described in our previous report with some modifi cations (4). An amount of 880 μl of 2 mM substrate (L- Dopa dissolved in 50 mM phosphate buffer, pH 6.8) was mixed with 100 μl of the tested sample (dissolved in DMSO) at 25°C for 2 min. Then, 20 μl of tyrosinase (1000 U/ml in phosphate buffer) was added to initiate the reaction. The assay mixture was incubated at 25°C for 10 min. The increase in absorbance at 475 nm due to the formation of dopa chrome was monitored with a spectrophotometer. The percentage of inhibition of tyrosinase activ- ity was calculated as follows: % inhibition = [(A − B)/A] × 100, where A is the absorbance at 475 nm with DMSO instead of the tested sample, and B is the absorbance at 475 nm with the sample. The concentration of a compound at which 50% of the enzyme activity was inhibited (the IC50 value) was obtained by linear curve fi tting. The isolated com- pounds’ inhibition kinetics was analyzed by the Lineweaver-Burk method. Experiments were carried out using the same protocol described above, except for the concentration of L-Dopa (0.2 to 1 mM). 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 The ethanol extract of the root of P. suffruticosa showed inhibitory activity against mush- room tyrosinase. Partition guided by the tyrosinase inhibitory activity indicated that the ethyl acetate and n-BuOH layer from the ethanol extract showed the bioactivity. Six com- pounds were purifi ed by silica gel column chromatography, LH-20, Lobar RP-8, and high-performance liquid chromatography from the ethyl acetate and n-BuOH layer. Their structures were elucidated with MS and NMR spectroscopic analysis by comparing the spectra data with those of previous reports (Figure 1). The mushroom tyrosinase inhibitory effects of the six compounds were evaluated using L-dopa as an enzyme substrate and kojic acid as a positive control. The results are shown