change the Km value, while the Vmax decreased from 0.308 to 0.006 μM/min (Figure 3(c)). Referring to the
Lineweaver-Burk plot, the inhibition mechanism of NANA on mushroom tyrosinase was classified as mixed
type I inhibition, whilst the inhibition mechanism of NANA on human tyrosinase was classified as non-competitive
inhibition. Enzymatic parameters of other known tyrosinase inhibitors were compared (Table 1). The enzymatic
parameters of inhibition by NANA were comparable to those existing known inhibitors.
In cultured B16 mouse melanoma cells, the color of culture medium was turned into dark brown after 48
hours of culture (Figure 4(a)). This phenomenon was due to formation and release of melanin-rich melanosome
from B16 melanoma [16]. For A375 human melanoma, after stimulated by α-MSH, culture medium was also
turned into dark brown after 48 hours of culture (Figure 4(c)) Vitamin C, tBHQ and 2% kojic acid, served as
positive control, successfully abolished the formation of melanin, and hence the medium color remained unchanged,
i.e. inhibition of melanin formation (Figure 4(a) and Figure 4(c)). The melanin concentration extracted
from the treated B16 mouse melanoma and A375 human melanoma also agreed with these observations.
The melanin concentration of cultured B16 and A375 were significantly lower than the blank control after incubation
with NANA and White or Red EBN extract; however, the abolishing effect was not found by the extract
of Grass EBN (Figure 4(b) and Figure 4(d)).
In 3D human skin model, treatment of 2% kojic acid successfully lightened the apparent intensity of skin color
(Figure 5(a)) and reduced the density of melanocytes (Figure 5(b)). Similar inhibitory results were obtained
by NANA and the extract of White EBN (Figure 5(a), Figure 5(b)). No significant differences in inhibition effects
between treatments on the sides of keratinocyte and melanocyte. Thus, kojic acid, NANA and EBN extracts
were all suggested to be permeable through the 3D human skin model (Figure 5(a), Figure 5(b)).