ANTIMELANOGENIC EFFECTS OF HMF 121 A Theophylline Figure 2. Inhibitory effects of HMF on the cellular melanogenesis stimulated by theophylline. B16F10 cells were pretreated with HMF or arbutin for 60 minutes and then stimulated with 1.0 mM theophylline for 72 hours. Cell pigmentation was observed under microscopy (A). Cell images shown are representative of at least three independent studies. The melanin content in the cell-free culture media is expressed as % of vehicle control (B and C). Data represent mean ± SEM (n = 3). *p 0.05 vs theophylline only. 3A, HMF attenuated the theophylline-dependent increase of tyrosine protein, suggest- ing that HMF might inhibit melanogenesis by involving down-regulation of the tyros- inase protein content. Arbutin had no inhibitory effect on the tyrosinase protein level (Figure 3B), as has been previously reported (18). HMF could have decreased the tyrosinase protein level either by inhibiting gene ex- pression or by increasing protein degradation. To address this issue, the next experiment examined whether HMF inhibited tyrosinase mRNA expression stimulated by theoph- ylline. Total cellular RNA was extracted from the treated cells and subjected to RT-PCR analysis for tyrosinase and housekeeping GAPDH mRNAs. The tyrosinase and GAPDH gene-specific primers designed in the present study successfully amplified the predicted PCR products of 564 bp and 396 bp, respectively, from the total cellular RNA, as shown in Figure 3C. HMF appeared to lower the tyrosinase mRNA level in a dose-dependent manner (Figure 3C) in agreement with its effects on the tyrosinase protein content (Figure 3A). The GAPDH mRNA level remained virtually constant (Figure 3C). There- fore the antimelanogenic effects of HMF could be attributed at least partly to the down-regulation of tyrosinase gene expression. Previous studies have shown that HMF scavenged 1,1-diphenyl-2-picrylhydrazyl free radicals (SC 50 = 3.0 µg/ml) and inhibited the autoxidation of linolenic acid (IC 9 0 = 32 µg/ml) more effectively than ascorbic acid (SC 50 = 3.3 µg/ml IC 90 = 124 µg/ml) (14). HMF also inhibited DOPA autoxidation (IC 50 = 60 µg/ml) and the catalytic activity of tyrosinase of mushroom origin (IC 50 = 100 µg/ml) more effectively than arbutin (IC50 300 µg/ml in both cases) (15). The current study is consistent with these previous findings. As shown in Figure 4A, HMF inhibited the tyrosinase-catalyzed melanin formation from DOPA in cell-free lysates (IC 50 = 50 µM) much more effectively than arbutin (IC50 1.6 mM).

122 JOURNAL OF COSMETIC SCIENCE II( :c C zC 0::: D. E ca: CD CJ ti) ca .,. C CD · .2: e !! , CD 1-.!:,. ·= c 2.0 B � ti 1.5 a.aa i .S 1.0 -� � en.:: e.!! , CD t- .!:.. 0.0 Tyrosinase &-actin 1.2 .----------------, 1.0 + + + 100 200 400 Tyrosinase (564bp) Figure 3. HMF down-regulates tyrosinase gene expression. Bl6Fl0 cells were pretreated with HMF or arbutin for 60 minutes and then stimulated with 1.0 mM theophylline for 72 hours. Cell lysates were analyzed for tyrosinase and 13-actin proteins by Western blotting (A and B). Blots shown are representative of at least three independent studies. Densitometry was performed to quantify the developed bands, and the graphs show the tyrosinase protein content relative to !3-actin. Total cellular RNA was extracted and subjected to RT-PCR analysis of tyrosinase and GAPDH mRNA (C). Stained gels shown are representative of at least three independent studies. M, size marker NC, negative control without RNA sample. The graphs show the tyrosinase mRNA content relative to GAPDH. Data represent mean ± SEM (n = 3). *p 0.05 vs theophylline only. The next experiments examined if HMF could inhibit the tyrosinase-catalyzed melanin formation from the DOPA in situ in the cells. For this purpose, cells were treated with DOPA after pretreatment with HMF or arbutin, in the absence of theophylline. As shown in Figure 4B, incubation of cells with DOPA alone resulted in the accumulation of a dark pigment inside the cells, providing a chance to determine the effect of test materials on the tyrosinase-catalyzed reactions in situ in the cells. As also shown in Figure 4B, the DOPA-dependent cell pigmentation was markedly attenuated by HMF. Arbutin showed a less significant effect, in agreement with the in vitro results. Therefore, it is concluded that HMF can interfere with tyrosinase-catalyzed reactions inside the cells. While genetic background is the most important factor for skin pigmentation (19), other non-genetic factors including hormonal change, chronic inflammation, aging, and ultraviolet light all affect skin pigmentation by stimulating the expression of tyrosinase and other enzymes involved in melanogenesis (2). Tyrosinase inhibitors may be the most non-invasive strategy for the control of skin pigmentation. However, they exhibit less consistent effects in vivo (11-13). The inhibitors of tyrosinase gene expression or matu-