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Vol. 51, No. 7
Table 3. Effects of a-Arbutin, Arbutin, b-Ab-a-G1 and b-Ab-a-G2 on
Human Tyrosinase
Compound
IC50 (mM)
Ki (mM)
a-Arbutin
2.1
Ͼ30.0
5.7
0.2
4.2
0.7
0.9
Arbutin
b-Ab-a-G1 (1)
b-Ab-a-G2 (2)
6.1
gested that a-arbutin was not a better skin-whitening agent
than arbutin. On the other hand, Funayama et al.6) reported
that a-arbutin strongly inhibited the tyrosinase from B16
mouse melanoma cells, while arbutin did not. According to
Fig. 4. Inhibitory Effects of a-Arbutin, Arbutin, b-Ab-a-G1 and b-Ab-a-
G2 on Tyrosinase from Human Malignant Melanoma
Tyrosinase activity was measured using 3.3 mM L-DOPA as the substrate. Results are our study described here, a-arbutin inhibited human tyrosi-
expressed as the percentage of inhibition by a-arbutin (᭺), arbutin (ᮀ), b-Ab-a-G1
nase much more strongly than arbutin. These results obtained
(᭹) or b-Ab-a-G2 (᭝) with respect to the untreated control.
by using the tyrosinase from mammalian cells indicated that
a-arbutin is a more effective skin-whitening agent than ar-
butin. Since mushroom tyrosinase is commercially available,
it might have been thought that the enzyme was useful for
the first screening of a tyrosinase inhibitor. However, the
amino acid sequence identity between human tyrosinase12)
and mushroom tyrosinase (Gene bank accession No.,
O42713) is only 23%. On the other hand, human tyrosinase
and murine tyrosinase13) are highly homologous (82% se-
quence identity). Therefore, it is definitely important to use
human tyrosinase for screening skin-whitening agents.
a-Arbutin is the strongest inhibitor of human tyrosinase
among the hydroquinone-glycosides which we studied so far.
Further investigation of the inhibitory effect of a-arbutin on
melanogenesis is now in progress.
Fig. 5. Lineweaver–Burk Plots Showing the Reciprocal of the Velocity
(1/V) of the Human-Tyrosinase Reaction vs. the Reciprocal of the Substrate
Concentration (1/S) in the Presence and Absence of the Inhibitor
Experimental
The rate of the tyrosinase reaction in the presence of 0.3 mM (ᮀ) or 0.5 mM (᭝) of a-
arbutin and in the absence of inhibitor (᭺). A475 represents an increase in the ab-
General Experimental Procedures Mass spectra were recorded on a
KRATOS Compact-Maldiseq instrument. 1H-NMR (500 MHz) and 13C-
NMR (125 MHz) spectra were obtained using a JEOL JNM-A500 spectrom-
eter in D2O containing [2,2,3,3-D4] sodium 3-3-(trimethylsilyl) propanoate
as an internal standard.
Chemicals Arbutin and L-DOPA were purchased from Sigma Chemical
Co. (St. Louis, MO, U.S.A.). CGTase from Bacillus macerance was pur-
chased from Amano Pharmaceutical Co., Ltd. (Nagoya, Japan). Other chem-
icals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan).
Preparation of a-Arbutin a-Arbutin was prepared by the method de-
scribed by Nishimura et al.5) a-Amylase from Bacillus sp. strain X-23 was
added to 100 ml of 50 mM sodium acetate buffer solution (pH 5.0) containing
5% hydroquinone and 20% maltopentaose. After incubation at 40 °C for
16 h, glucoamylase from Aspergillus niger was added, and the mixture was
incubated at 40 °C for 4 h. a-Arbutin was purified from the reaction mixture
by extraction with ethylacetate and charcoal column chromatography.
Preparation of Tyrosinase from Human Malignant Melanoma
Human malignant melanoma cells, HMV-II,10) were provided by the Cell Re-
source Center for Biomedical Research, Tohoku University. The cells were
cultured in F12/DMEM supplemented with 10% fetal bovine serum and
100 mM L-DOPA at 37 °C in a humidified atmosphere of 5% CO2 in air. On
the 10th day, the cells were scraped out from the tissue culture plate with
Mg2ϩ-, Ca2ϩ-free phosphate-buffered saline [PBS(Ϫ)], and were homoge-
nized in PBS(Ϫ) with a glass homogenizer at 4 °C. And the homogenate was
fractionated by differential centrifugation based on the method of
Claude.14,15) The homogenate was centrifuged at 1000ϫg for 10 min. The
precipitate was sonicated in PBS(Ϫ) on ice, and the mixture was centrifuged
at 10000ϫg for 30 min. The supernatant containing tyrosinase was used for
the measurement of the inhibitory effects.
sorbance at 475 nm.
but weaker than a-arbutin (Fig. 4). The IC50 values of b-Ab-
a-G1 and b-Ab-a-G2 were 5.7 mM and 6.1 mM, respectively
(Table 3). According to Lineweaver-Burk plots of the tyrosi-
nase activity, the inhibition of a-arbutin against human ty-
rosinase was indicated to be competitive, as shown in Fig. 5.
Based on these kinetic data, the Ki value for a-arbutin was
calculated as 0.2 mM (Table 3). Similar kinetic analyses indi-
cated that the inhibitions of b-Ab-a-G1 and b-Ab-a-G2
were also competitive. The Ki values for b-Ab-a-G1 and b-
Ab-a-G2 and arbutin were 0.7 mM, 0.9 mM and 4.2 mM, re-
spectively (Table 3). The Ki values for b-Ab-a-G1 and b-Ab-
a-G2 were about 6-fold lower than that for arbutin. Jergil et
al.11) reported that the KM value for human tyrosinase was
0.5 mM when L-DOPA was used as the substrate, but 3 mM
when D-DOPA was used as the substrate. It would be interest-
ing to examine whether the difference in inhibitory activity
of a-arbutin and arbutin is correlated with the high stereo
specificity of human tyrosinase. We are now in the process of
synthesizing a-arbutin-a-glycosides and analyzing their ef-
fect on human tyrosinase activity.
The inhibitory effects of a-arbutin and arbutin have been
previously compared.6,9) Nishimura et al.9) reported that a-
arbutin, as well as arbutin, inhibited mushroom tyrosinase.
Funayama et al.6) reported that a-arbutin did not inhibit
Assay of Tyrosinase Activity Tyrosinase activity was assayed according
to the method of Funayama et al.16) with slight modification. The reaction
mixture (90 ml) contained 3.3 mM L-DOPA in 0.33 M phosphate buffer (pH
mushroom tyrosinase, while arbutin did. These works sug- 7.0) and the enzyme in the presence or absence of inhibitors. Thirteen units