Inhibitory effects of 5,6,7-trihydroxyflavones on tyrosinase

Article abstract of DOI:10.3390/12010086

Baicalein (1), 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-hydroxykaempferol (14), which are naturally occurring flavonoids from a set of 14 hydroxyflavones tested, exhibited high inhibitory effects on tyrosinase with respect to L-DOPA, while each of the 5,6,7-trihydroxyflavones 1, 6, 13 or 14 acted as a cofactor to monophenolase. Moreover, 6-hydroxykaempferol (14) showed the highest activity and was a competitive inhibitor of tyrosinase compared to L-DOPA. 5,6,7-Trihydroxyflavones 1, 6, 13 or 14 showed also high antioxidant activities. Hence, we conclude that the 5,6,7-trihydroxy-flavones are useful as good depigmentation agents with inhibitory effects in addition to their antioxidant properties.

Full text of DOI:10.3390/12010086

Molecules 2007, 12, 86-97
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Inhibitory Effects of 5,6,7-Trihydroxyflavones on Tyrosinase
Hong Gao, Jun Nishida, Shizuka Saito and Jun Kawabata*
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan
* To whom correspondence should be addressed. Tel: +81-11-706-2496. Fax: +81-11-706-2496;
E-mail: junk@chem.agr.hokudai.ac.jp
Received: 8 December 2006; in revised form: 24 January 2007 / Accepted: 24 January 2007 /
Published: 29 January 2007
Abstract: Baicalein (1), 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-hydroxy-
kaempferol (14), which are naturally occurring flavonoids from a set of 14 hydroxy-
flavones tested, exhibited high inhibitory effects on tyrosinase with respect to L-DOPA,
while each of the 5,6,7-trihydroxyflavones 1, 6, 13 or 14 acted as a cofactor to
monophenolase. Moreover, 6-hydroxykaempferol (14) showed the highest activity and
was
a
competitive inhibitor of tyrosinase compared to L-DOPA. 5,6,7-
Trihydroxyflavones 1, 6, 13 or 14 showed also high antioxidant activities. Hence, we
conclude that the 5,6,7-trihydroxy-flavones are useful as good depigmentation agents
with inhibitory effects in addition to their antioxidant properties.
Keywords: Tyrosinase inhibitor; flavonoids; 5,6,7-trihydroxyflavones; baicalein;
melanin; depigmentation.
Introduction
Melanin is the general term assigned to a group of phenolic pigments produced from tyrosine and
related compounds by an oxidative polymerization. Tyrosinase (EC 1. 14. 18. 1), widely distributed in
Nature, is a copper-containing enzyme that catalyzes two different reactions: a hydroxylation of
monophenols to o-diphenols (monophenolase activity) and an oxidation of o-diphenols to o-quinones
(diphenolase activity), both using molecular oxygen [1]. It is well known that tyrosinase is a key
enzyme for melanin biosynthesis in plants and animals. Therefore, tyrosinase inhibitors have been

Molecules 2007, 12
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reported and tested as therapeutic agents for the treatment of melanin hyperpigmentation and as
cosmetic materials for whitening after sunburn [2]. Melanin formation is also considered to be
deleterious to the color quality of fruits and vegetables, so an inhibition of this enzyme should be
useful for browning control of plant-derived foods in the food industry [3]. Furthermore, tyrosinase is
one of the most important key enzymes in insect molting process, and investigating its inhibitors may
be important in finding alternative insect control agents [4,5].
Many efforts have been addressed to the search for effective and safe tyrosinase inhibitors from
natural materials, and flavonoids are one of the most numerous and best-studied group of plant
polyphenols. It is reported that some flavonoids inhibited tyrosinase activity by active chelation and
others acted as cofactors and/or substrates towards tyrosinase [6-9]. Baicalein, 5,6,7-trihydroxyflavone
(1), originally isolated from Scutellaria baicalensis, has reportedly been used as a depigmentation
agent in cosmetics [10,11]. In the previous study, the inhibitory effects by 1 on tyrosinase and melanin
formation in skin were evaluated, but the structure-activity relationship of 1 towards tyrosinase
remains unknown.
Baicalein (1, Figure 1) is a unique flavone bearing a 6-hydroxyl group together with the standard
5,7-dihydroxyl substituents, and it shows a variety of biological and pharmacological effects including
antioxidant [12], antimutagenic [13], anti-inflammatory [14], antimicrobial [15], and anticarcinogenic
properties [16]. We previously reported the inhibitory effect and mechanism of the interaction of 1 and
related 5,6,7-trihydroxyflavones towards rat intestinal α-glucosidase [17-21]. As a part of our research
on the new biological and physiological properties of 6-hydroxyflavones, we are interested in the
inhibitory effect on melanin formation by 1 and its related compounds. In order to evaluate the
inhibitory effect on melanin biosynthesis by 5,6,7-trihydroxyflavones, we synthesized a series of
natural hydroxyflavones (Figure 1) and conducted the structure-activity relationship study for their
tyrosinase inhibition, together with demonstrating the inhibitory mechanism in the present study. In
addition, we evaluated their antioxidant potency, which can contribute to prevent pigmentation due to
non-enzymatic auto-oxidation.
Results and Discussion
Inhibitory effects on L-DOPA oxidizing diphenolase activity of tyrosinase of baicalein (1) and
related hydroxyflavones 2-14 are summarized in Table 1. At a concentration of 400 µM, 1 inhibited
69% of tyrosinase activity with respect to L-DOPA as a substrate, the where 50% inhibitory
concentration (IC50) was calculated to be 273 µM. On the other hand, 4, which lacks a 6-hydroxyl
group, did not show any activity. Hence, this result indicates that the 6-hydroxyl substituent of 1 is an
important factor for the tyrosinase inhibition activity. Further evidence supporting this rationale is that,
compounds 9-11, all of which do not possess 6-hydroxyl group, showed no activity; however, 6, 7 and
8, all of which possess 6-hydroxyl group, showed a higher activity than their unsubstituted
counterparts, 9, 10 and 11. Furthermore, removal of any hydroxyl group at positions 5,6,7 (eg. 2 and 3)
led to a dramatic loss of potency, and compounds 9-11 having no 5,6,7-trihydroxyflavone structure
also did not inhibit tyrosinase activity. Hence, these results indicate that all of the three hydroxyl
substituents of 1 are important for an inhibition of the tyrosinase activity.

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Figure 1. Chemical structures of baicalein (1) and related hydroxyflavones 2-14.
Table 1. Mushroom tyrosinase inhibitory activities of hydroxyflavones (1-14).
compound
IC50 (µM)
diphenolasea
monophenolaseb
1
2
273
NIc
cofactor
NT
3
NI
NT
4
NI
NT
5
NI
NT
6
193
cofactor
NT
7
400 (38%)
400 (43%)
NI
8
NT
9
NT
10
11
12
13
NI
NT
400 (33%)
262
NT
cofactor
NT
182
14
124
cofactor
a
With respect to L-DOPA.
b
c
With respect to L-tyrosine.
Less than 10% inhibition at the concentration of 400 µM. NT, not tested. Cofactor, the lag time
was shortened or eliminated by the presence of the test sample.

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Having identified the importance of the 5,6,7-trihydroxyl group of 1, we decided to explore the
incorporation of an additional hydroxyl group onto position 8 of compound 1 to afford 5.
Unexpectedly, this led to a reduction in potency. Among the B-ring-hydroxylated 5,6,7-
trihydroxyflavones, it is interesting to note that the highest activity was observed for
monohydroxylated 6 (IC50=193 µM), compared to dihydroxylated 7 (38% inhibition at the
concentration of 400 µM) and trihydroxylated 8 (43% inhibition at the concentration of 400 µM).
Hence, it was suggested that the enhanced inhibitory activity of 1 is solely due to the 4’-hydroxylation
of the B-ring, and extra hydroxyl group substituents of the A- or B-rings of 1 are not always
advantageous. It is unclear, however, why the additive effect of the 4’-OH is different from that of the
other hydroxyl substitution.
Kubo and coworkers have reported the diphenolase-inhibitory activity of flavonols, and indicated
the importance of a hydroxyl group at position 3 [6]. In order to estimate the effect on the inhibitory
activity against diphenolase of the introduction of a 3-hydroxyl group in 1, flavonols 12-14 were tested
(Table 1). Compounds 13 (IC50=182 µM) and 14 (IC50=124 µM) showed higher activity than that of 1,
while even 12 (IC50=262 µM) showed the comparable activity to 1, which validates the importance of
the 3-hydroxyl group of 1 for the activity as well as the basic 5,6,7-trihydroxyflavone structure.
Having demonstrated the diphenolase-inhibitory activity of 5,6,7-trihydroxyflavones 1, 6, 13 and
14, we then evaluated their L-tyrosine oxidizing monophenolase activity of mushroom tyrosinase.
Monophenolase activity of tyrosinase is characterized by a lag time, derived from oxidative
hydroxylation of monophenolic substrates to o-diphenols. As shown in Figure 2, the lag time was not
observed for 1, 6, 13 or 14. It was reported that quercetin (o-diphenols) acted as an alternative cofactor
to initiate monophenolase activity [7]. Judging from the structure of 1, 6, 13 or 14 (which possesses
5,6,7-trihydroxyflavone structure), we can conclude that 1, 6, 13 or 14 are co-factors to tyrosinase
monophenolase.
Figure 2. Lag period of L-dopaquinone formation. L-tyrosine (0.8 mM) was incubated
with tyrosinase in the absence (control) or with the addition of baicalein (1, 320 µM), 6-
hydroxyapigenin (6, 200 µM), 6-hydroxygalangin (13, 320 µM) and 6-
hydroxykaempferol (14, 100 µM).

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Among the tested hydroxyflavones 1-14, compound 14 showed the highest diphenolase-inhibitory
activity, and hence, we carried out the steady-state kinetic analysis of 14 for tyrosinase inhibition with
respect to L-DOPA as a substrate. Lineweaver-Burk plots for the inhibition of tyrosinase (diphenolase)
by 14 were obtained with variable concentrations of 14 and the substrate (Figure 3). The intersection
of these lines on the vertical axis indicated that 14 was a competitive inhibitor of tyrosinase with
respect to L-DOPA as a substrate, with a Ki value of 148 µM. These data strongly suggested that 14
effectively inhibited the activity by binding to the active site of this enzyme.
Figure 3. Lineweaver-Burk plots for the inhibition of tyrosinase by 6-hydroxykaempferol
(14) with respect to L-DOPA as substrate.
In previous studies, flavonoids were reported to form a chelate with the copper in tyrosinase and
thus irreversibly inactivate this enzyme [6,7]. In particular, it has been reported recently that the ability
of flavonoids to form the chelate with copper ions plays an important role in the tyrosinase inhibition
[22]. To further confirm the inhibitory mechanism of 14, the copper-ion chelating ability of 14 was
examined. A solution of 14 (50 µM) in phosphate buffer was scanned with or without CuSO4 (100
µM), and the spectra obtained was compared (data not shown). It was observed that the characteristic
absorption maxima at 272 nm and 359 nm shifted to 287 nm and 386 nm, respectively, with the
addition of copper ion; and returned to their original values 272 nm and 359 nm of the addition of 250
µM EDTA. This copper chelating ability can be further supported by the observation of a noticeable
shift (358 →303 nm) when 14 was incubated with enzyme. The characteristic hypsochromic shift was
observed in the absorption spectrum of 14 and tyrosinase complex, similar to that observed in the other
flavonols [7], such as quercetin. These results suggested that the mechanism of inhibition by 14 could
be due to its chelation with copper ion in the active site of the enzyme so as to irreversibly inactivate
the enzyme, although the detailed three-dimensional structure of the mushroom tyrosinase and the
binding-model of 14 with this enzyme blocked.

Molecules 2007, 12
Table 2. ABTS radical scavenging activity and superoxide dismutase (SOD)-like activity
91
of baicalein (1), 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-hydroxy-
kaempferol (14).
Compounds
EC50 (µM)
ABTS radical scavenging activity
SOD-like activity
1
4.89
4.43
3.33
2.59
10.86
NT
48.6
36.7
46.9
34.3
NT b
42.5
6
13
14
Trolox®,a
L-Ascorbic acid c
a Trolox® is used as the positive control of ABTS radical scavenging activity.
b Not tested.
c L-Ascorbic acid is used as the positive control of SOD-like activity.
Melanin biosynthesis is caused by the production of dopachrome from dopa catalyzed by tyrosine
and concurrent auto-oxidative polymerization of dopachrome [1]. Moreover, compounds with reducing
properties can have depigmentation effects by interaction with o-quinones, thus avoiding the oxidative
polymerization of melanin intermediates [1]. In addition, these compounds contain phenolic hydroxyls,
which can prevent pigmentation resulting from auto-oxidative processes [23,24]. Therefore, we tested
the antioxidant ability of 5,6,7-trihydroxyflavones 1, 6, 13 and 14 by measuring the ABTS radical
scavenging as well as superoxide dismutase (SOD)-like activity, closely related to the auto-oxidation.
ABTS radical scavenging activity (EC50) of 1, 6, 13 and 14 ranged from 2.6 µM to 4.9 µM, which is
higher than that of Trolox® (EC50=10.9 µM) as a positive control (Table 2). Superoxide dismutase
-
(SOD)-like activity was estimated by suppression of the superoxide anion radical (O2 ) generated from
the hypoxanthine-xanthine oxidase system in the presence of test samples. As shown in Table 2, 5,6,7-
trihydroxyflavones 1, 6, 13 and 14 exhibited strong SOD-like activity with EC50s at the order of 10-5 M,
which is comparable to that of L-ascorbic acid as a positive control. These results indicate that the
5,6,7-trihydroxyflavones may contribute to the inhibition of melanin biosynthesis in cells by their
potent antioxidant activities.
Conclusions
We have disclosed considerable information about the structure-activity relationships with respect
of the number and relative position of the hydroxyl groups of baicalein (1) for enhancing the inhibitory
activity against tyrosinase, and found that 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-
hydroxykaemferol (14) all exhibited high inhibitory effects towards tyrosinase. Moreover, 14 showed
the highest activity among the tested flavonoids, and 14 was a competitive inhibitor of tyrosinase with
respect to L-DOPA. 5,6,7-Trihydroxyflavones 1, 6, 13 and 14 also showed high antioxidant activities.
Hence, we may conclude that the 5,6,7-trihydroxyflavones are useful as good depigmentation agents
with inhibitory effects on tyrosinase in addition to their antioxidant properties.

Molecules 2007, 12
Experimental Section
General
92
13
NMR spectra were recorded with a Bruker AMX500 (1H-, 500 MHz; C-, 125 MHz) instrument.
Chemical shifts are expressed in ppm referenced to the residual solvent signals of methanol-d4 (δH 3.30
ppm), chloroform-d (δH 7.24 ppm) and dimethyl sulfoxide-d6 (δH 2.49 and δC 39.5 ppm). Coupling
constants (J) are reported in Hz. Fast atom bombardment (FAB), Field desorption (FD), FD-high
resolution (HR), electron ionization (EI), and EI-HR mass spectra (MS) were obtained on a Jeol JMS-
SX102A instrument. Melting points were measured on a hot stage and are uncorrected. The following
experimental conditions were used for chromatography: normal phase column chromatography: Silica
gel Wakogel C-300 (Wako Pure Chem. Co., Osaka, Japan, 40-64 mesh), reverse phase ODS column
chromatography; Cosmosil 75C18-OPN (Nacalai Tesque, Inc., Kyoto, Japan). TLC, precoated TLC
plates with Silica gel 60 F254 (Merck, 0.25 mm or 0.5 mm thickness, normal phase) and Silica gel RP-
18 F254s (Merck, 0.2 mm thickness, reverse phase). Detection was done with a UV lamp (254 nm).
Preparative HPLC was done with an Inertsil PREP-ODS column (20.0×250 mm, GL-Science). The
appropriate analytical conditions are mentioned in each section. All reagents were of reagent grade and
were purchased from Wako Pure Chem. Co., Osaka, Japan, unless otherwise stated. Acetone and
tetrahydrofuran were dried by storage over 3Å molecular sieves. All solvents were distilled before use.
All nonaqueous reactions were done in dry glassware.
Test Compounds
Among the tested hydroxyflavones 1-14 (Figure 1), baicalein (1), 5,6-dihydroxyflavone (2), 6,7-
dihydroxyflavone (3), 5,6,7,8-tetrahydroxyflavone (5), 6-hydroxyapigenin (6), 6-hydroxylutelin (7),
3’,4’,5’,5,6,7-hexahydroxyflavone (8) and 6-hydroxygalangin (13) were synthesized as described
previously [19-21]. 5,7-Dihydroxyflavone (4) was purchased from Tokyo Kasei Kogyo Co., Tokyo,
Japan. Apigenin (9), luteolin (10) and kaempferol (12) were purchased from Sigma Aldrich Japan
Co.,Tokyo, Japan. 3’,4’,5,5’,7-Pentahydroxyflavone (11) and 6-hydroxykaempferol (14, Scheme 1)
were synthesized as described below.
3’,4’,5,5’,7-Pentahydroxyflavone (11).
A mixture of 5,7-dihydroxy-3’,4’,5’-trimethoxyflavone (112 mg, 0.33 mmol) [20], 47% aqueous
hydrobromic acid (1.1 mL, 9.9 mmol) and acetic acid (4 mL) was heated under reflux for 23 h . After
cooling to room temperature, the reaction mixture was extracted with 1-butanol and concentrated to
dryness. The residue was dissolved in a small amount of methanol and subjected to reverse-phase
column chromatography, using water-methanol-formic acid (40:60:0.1) as the eluent to yield crude 11.
This was further purified by preparative HPLC (mobile phase, water-methanol-formic acid
(40:60:0.1); flow rate, 5.0 mL/min; detection, UV 254 nm) to give 11 (tR 21 min, 35 mg, 38%) as pale
1
yellow powders: mp >300 °C; FAB-MS (negative) m/z (%) 301 ([M-H]-, 100); H-NMR δ (DMSO-

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d6): 6.17 (1H, d, J=1.6, 6-H), 6.39 (1H, d, J=1.6, 8-H), 6.52 (1H, s, 3-H), 6.95 (2H, s, 2’ and 6’-H),
12.96 (1H, s, 5-OH).
Synthesis of 6-hydroxykaempferol (14): 6’-Hydroxy-2’,3’,4,4’-tetramethoxychalcone (14a).
To 6’-hydroxy-2’,3’,4’-trimethoxyacetophenone [25] (6.1 g, 26.6 mmol) and 4-methoxy-
benzaldehyde (3.3 mL, 27 mmol) in ethanol (32 mL) was added 50% KOH (15 g) at 0 °C and the
mixture was then stirred under argon gas at room temperature for 32 h. The reaction was quenched
with ice water (300 g) and acidified with 6M HCl. The resulting mixture was extracted with ethyl
acetate (300 mL x 3) and the combined organic extracts were washed with brine, dried over anhydrous
Na2SO4 and concentrated. The residue was purified by silica gel column chromatography using
hexane-ethyl acetate (8:2) as the eluent to give 14a (8.1 g, 89%) as a pale yellow powder: mp 137-139
1
°C (lit. [25] mp 139-141°C); FDHRMS m/z 344.1235 (calcd. for C19H20O6, 344.1260); H-NMR δ
(chloroform-d): 3.82, 3.85, 3.89, 3.91 (each 3H, 2’,3’,4,4’-OMe), 6.28 (1H, s, 5’-H), 6.93 (2H, d,
J=8.9, 3 and 5-H), 7.59 (2H, d, J=8.9, 2 and 6-H), 7.82-7.84 (2H, m, α and β-H), 13.75 (1H, s, 2’-OH).
Scheme 1. Synthesis of 6-hydroxykaempferol (14).
Reagents and conditions: (i) Ac2O, AcONa, 110 °C, 3 h; (ii) BF3-Et2O, 70 °C, 2 h; (iii) 4-
methoxybenzaldehyde, EtOH, KOH, R.T, 36 h; (iv) I2, DMSO, 190-200 °C, 2h; (v) LiN(i-Pr)2,
(MeO)3B, AcOH, 20% H2O2, THF; (vi) BBr3, CH2Cl2, -80 °C for 1 h and R.T for 12 h.
4’,5,6,7-Tetramethoxyflavone (14b)
A mixture of 14a (7.3 g, 21.2 mmol) and iodine (250 mg) in DMSO (25 mL) was heated at 190 –
200 °C for 2 h, and then poured onto ice water (300 g). This mixture was partitioned with ethyl acetate
and water. The organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography using hexane-ethyl acetate (7:3) as the

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eluent to give 14b (5.4 g, 74%) as a pale yellow powder: mp 144-146 °C (lit. [26] mp 142 °C);
1
FDHRMS m/z 342.1094 (calcd. for C19H18O6, 342.1103); H-NMR δ (DMSO-d6): 3.75, 3.79, 3.84,
3.94 (each 3H, 4’,5,6,7-OMe), 6.70 (1H, s, 3-H), 7.08 (1H, dd, J=6.8, 2.3, 3’ and 5’-H), 7.20 (1H, s, 8-
H), 8.01 (2H, dd, J=6.8, 2.3, 2’ and 6’-H).
3-Hydroxy-4’,5,6,7-tetramethoxyflavone (14c)
n-Butyllithium (1.6 M solution in hexane, 5 mL, 8 mmol) was added to diisopropylamine (1.2 mL, 8
mmol) in dry THF (40 mL) at –80 °C under argon gas. The resulting lithium diisopropylamide (LDA)
solution was warmed to 0 °C for 30 min and re-cooled to –80 °C. Compound 14b (1.4 g, 4 mmol) in
THF (40 mL) was added dropwise to the LDA solution and stirred for 1 h. After an addition of
trimethyl borate (0.9 mL, 8 mmol) in THF (20 mL), the resulting solution was then stirred at –80 °C
for 30 min. The reaction mixture was acidified with acetic acid (0.7 mL), stirred for 15 min, and then
oxidized with 30% hydrogen peroxide (1.2 mL, 8 mmol). The solution was allowed to warm slowly to
room temperature and was then shaken with aqueous saturated sodium bicarbonate solution (50 mL).
The aqueous mixture was extracted with ethyl acetate (100 mL x 3). The combined organic extracts
were dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column
chromatography using chloroform-methanol (10:0.2) as the eluent to give 14c (0.5 g, 35%) as a yellow
powder: mp 132-134 °C (lit. [27] mp 131-132 °C); FDHRMS m/z 358.1068 (calcd. for C19H18O7,
1
358.1052); H-NMR δ (DMSO-d6): 3.31, 3.76, 3.84, 3.93 (each 3H, s, 4’,5, 6 and 7-OMe), 7.10 (2H,
d, J=9.1, 3’ and 5’-H), 7.16 (1H, s, 8-H), 8.16 (2H, d, J=9.1, 2’ and 6’-H), 9.08 (1H, s, 3-OH).
6-Hydroxykaempferol (14).
To a stirred solution of 14c (358 mg, 1 mmol) in dry CH2Cl2 (10 mL) at -80 °C was added BBr3 (1M
CH2Cl2 solution, 8 mL, 8 mmol). The reaction mixture was then stirred for 1 h at –80 °C and 12 h at
room temperature. The reaction was stopped by adding ice-cooled water (50 mL). To this mixture was
added 1-butanol (200 mL). The organic phase was separated, washed with brine, and evaporated to
dryness. The residue was dissolved in a small amount of methanol and subjected to reverse-phase
column chromatography, using water-methanol-formic acid (40:60:0.1) as the eluent to yield crude 14.
This was further purified by preparative HPLC (mobile phase, water-methanol-formic acid
(30:70:0.1); flow rate, 4.0 mL/min; detection, UV 254 nm) to give 14 (tR 14.9 min, 176 mg, 58%) as a
pale yellow powder: mp 284-286 °C (lit. [28] mp 280-282 °C); FDHRMS m/z 302.0452 (calcd. for
C15H10O7, 302.0426); 1H-NMR δ (DMSO-d6): 6.51 (1H, s, 8-H), 6.91 (2H, d, J=8.9, 3’ and 5’-H), 8.02
(2H, d, J=8.9, 2’ and 6’-H).
Tyrosinase inhibition assays
Tyrosinase inhibition assays were performed according to a modified method described by Kubo
[6]. Mushroom tyrosinase (EC 1. 14. 18. 1, Sigma Product T3824 with an activity of 3320 units/mg)
was used for the bioassay in this study. The tested compounds were first dissolved in DMSO and
diluted 25 times with water in each experiment before use. The activity was expressed as the sample

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concentration that gave a 50% inhibition in the enzyme activity (IC50). For the measurement of
diphenolase inhibition, a sample solution (0.1 mL) and 1.5 M L-DOPA solution (1.5 mL, 0.1M
phosphate buffer, pH 6.8) were mixed with H2O (0.4 mL) and preincubated at 25 °C for 5 min. Then,
tyrosinase solution (0.5 mL, 125 units) was added and the formation of dopachrome was monitored at
475 nm from 0.5 to 4 min using a Hitachi UV-3210 spectrophotometer. For the measurement of
monophenolase inhibition, a sample solution (0.1 mL) and 2 M L-tyrosine solution (1 mL) were mixed
with of 0.1 M phosphate buffer (0.9 mL, pH 6.8), and pre-incubated at 25 °C for 5 min. Then,
tyrosinase solution (0.5 mL, 250 units) was added and the dopachrome formed was monitored at 475
nm for 20 min.
Chelation ability of 5,6,7-trihydroxyflavones towards copper ions
A solution of test compounds (10 mM) was prepared in DMSO. Then 50 µM solution was prepared
in a cuvette containing phosphate buffer (0.1 M, pH 6.8) and the sample absorbance was recorded in
the 200-540 nm range using Shimadzu UV-1600 spectrophotometer. A repeated scan was recorded
after addition of 100 µM CuSO4 and a third spectrum after addition of 250 µM EDTA. To check if
5,6,7-trihydroxyflavones can chelate copper in the enzyme, the UV-visible spectra (200-540 nm) was
measured. A mixture containing 0.1 M phosphate buffer (1.5 mL, pH 6.8), H2O (0.4 mL), a test
solution (0.1 mL) and tyrosinase solution (0.5 mL, 125 units/mL), was incubated at 25 °C for 30 min,
and then the spectra was recorded.
ABTS radical scavenging activity
ABTS radical scavenging activity was determined according to a modification of the method of
reference [29]. In brief, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt,
ABTS, 19 mg) was reacted with potassium persulfate (3.3 mg) overnight in the dark at room
temperature. The working solution was prepared by diluting it with water to get an absorbance around
0.70 at 734 nm. Test sample (30µL in DMSO) was reacted with diluted ABTS (2.97 mL) and
absorbance at 734 nm was recorded within 30 min. Trolox® was used as a positive control, and the
control was prepared without the samples. The activity was expressed as the concentration of sample
necessary to give a 50% reduction in the original absorbance (EC50).
SOD (superoxide dismutase)-like activity
SOD (superoxide dismutase)-like activity was measured by the nitrite method [24,30]. In brief, a
mixture of the sample solution (0.01 mL), EDTA-phosphate buffer solution (pH 8.2, 0.6 mL), reagent
A (0.5 mM xanthine and 10 mM hydroxylammonium chloride in buffer, 0.2 mL) and xanthine-oxidase
solution (10 mU/mL in buffer) was incubated at 37 °C for 30 min, and then coloring reagent was
added (25 µM N-(1-naphthyl)ethylenediamine, 2 mM sulfanilic acid and 16.7% acetic acid, 2 mL).
The resulting mixture was allowed to stand for 30 min at room temperature and the optical absorbance
was measured at 550 nm. SOD-like activity was expressed as a value of EC50 against the production of
superoxide anion from hypoxanthine-xanthine-oxidase.

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Acknowledgements
We are grateful to Mr. Kenji Watanabe and Dr. Eri Fukushi, GC-MS & NMR Laboratory, Faculty
of Agriculture, Hokkaido University, for their skillful mass spectral measurements.
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