INHIBITORY EFFECT OF GASTRODIA ELATA EXTRACT ON MELANOGENESIS 93 value, *p 0.05). In addition, there was a reduction in mRNA from either TRP-1 (45.2 ± 12.3% of control value, *p 0.05) or TRP-2 (38.6 ± 15.9% of control value, *p 0.05) (Figure 2B) compared to the control (Table II). EFFECT OF GE EXTRACT ON THE EXPRESSION OF TYROSINASE, TRP-1, AND TRP-2 PROTEINS To confi rm the inhibitory effect of GE extract on the expression of tyrosinase, TRP-1, and TRP-2 proteins, western blotting was performed to measure protein expression with β-actin as an internal control. Treatment of GE extract on HM3KO cell signifi cantly re- duced tyrosinase protein expression compared to the control (26.1 ± 10.1% of control value, *p 0.05). Moreover, the levels of both TRP-1 (60.6 ± 7.9% of control value, *p 0.05) and TRP-2 (35.8 ± 12.8% of control value, *p 0.05) proteins were signifi - cantly reduced compared to the control (Figures 3A and 3B). These results were in con- cordance with the mRNA expression results (Table II). DISCUSSION Mammalian pigmentation results from the synthesis and accumulation of photoprotec- tive epidermal melanins that are formed from the amino acid precursor l-tyrosine within specialized cells (22). Melanin, which determines the color of the skin and hair, transfers from melanocytes to keratinocytes after synthesis in the melanosomes (23). Although melanin plays an important protective role against UV light, overproduction and accu- mulation of melanin pigment could create serious skin problems such as freckles, age spots, and melasma (24). The inhibition of melanogenesis has been a matter of concern in the development of medicinal and cosmetic treatments for skin depigmenting and light- ening. The major mechanism of antimelanogenic action is direct inhibition of tyrosinase activity or inhibition of its gene expression. Other mechanisms include microphthalmia- associated transcription factor inhibition, downregulation of melanocortin receptor 1 activity, and interference with melanosome maturation and transfer. Another mechanism is increasing melanocyte loss via desquamation and chemical peeling. The inhibition of tyrosinase activity is the most common approach to achieve skin hypopigmentation, since Figure 1. Mushroom tyrosinase activity was decreased signifi cantly when treated with GE extract (69.7 ± 7.2% of control value, *p 0.05).

JOURNAL OF COSMETIC SCIENCE 94 this enzyme catalyzes the rate-limiting step of pigmentation. Human melanocytes are known to express TRP-1 and TRP-2 as well as tyrosinase (25–27). These proteins consti- tute a specifi c family of membrane proteins that are structurally related, but with distinct enzyme functions (28). TRP-2 functions as a DOPAchrome tautomerase and catalyzes the rearrangement of DOPAchrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), whereas TRP-1 oxidizes DHICA to a carboxylated indole-quinone. Both TRP enzymes Figure 2. The inhibitory effect of GE extract on the expression of tyrosinase, TRP-1, and TRP-2 mRNA. (A) The tyrosinase gene family mRNA was measured by RT-PCR. (B) Treatment of HM3KO cells with GE extract signifi cantly reduced tyrosinase mRNA expression compared to the control (43.8 ± 17.4% of control value, *p 0.05). Moreover, there was reduction in mRNA from either TRP-1 (45.2 ± 12.3% of control value, *p 0.05) or TRP-2 (38.6 ± 15.9% of control value, *p 0.05) compared to the control. Table II mRNA and Protein Expression of Tyrosinase, TRP-1, and TRP-2 in HM3KO Melanoma Cells Treated with GE Extract Compared to the Control mRNA expression by RT-PCR (% of control value) Protein expression by western blotting (% of control value) Tyrosinase 43.8 ± 17.4* 26.1 ± 10.1* TRP-1 45.2 ± 12.3* 60.6 ± 7.9* TRP-2 38.6 ± 15.9* 35.8 ± 12.8* *p 0.05.