Tyrosinase inhibitory polyphenols from roots of Morus Ihou

Article abstract of DOI:10.1021/jf8033286

Twelve polyphenols (1-12) possessing tyrosinase inhibitory properties were isolated from the methanol (95%) extract of Morus Ihou. The isolated compounds consisted of four flavanones (1 -4), four flavones (5-8), and four phenylbenzofuranes (9-12). Moracin derivative 12 proved to be new a compound which was fully characterized. Compounds 1-12 were evaluated for both monophenolase and diphenolase (the two steps catalyzed by tyrosinase) inhibition to identify the structural characteristics required for mushroom tyrosinase inhibition. We observed that all parent compounds (1, 5, and 9) possessing an unsubstituted resorcinol group were highly effective inhibitors of monophenolase activity (IC₅₀ values of 1.3, 1.2, and 7.4 μM). The potency of the inhibitors diminished with alkyl substitution on either the aromatic ring or the hydroxyl functions. Interestingly, flavone 5 was shown to possess only monophenolase inhibitory activity, but flavanone 1 and phenylbenzofuran 9 inhibited diphenolase as well as monophenolase significantly. The inhibitory mode of these species was also dependent upon the skeleton: phenylbenzofuran 9 manifested a simple competitive inhibition mode for monophenolase and diphenolase; on the other hand flavanone 1 (monophenolase, K₃ = 0.1966 min^-1 μM^-1 k₄= 0.0082 min ^～1, and K_i^app = 0.0468 μM; diphenolase, k₃ = 0.0014 min^-1 μM^-1 k₄ = 0.0013 min^-1, and K_i^app = 0.8996 μM) and flavone 5 both showed time-dependent inhibition against monophenolase. Compound 1 operated according to the simple reversible slow binding model whereas compound 5 operated under the enzyme isomerization model.

Full text of DOI:10.1021/jf8033286

J. Agric. Food Chem. 2009, 57, 1195–1203 1195
Tyrosinase Inhibitory Polyphenols from Roots of
Morus lhou
†
,‡
†,‡
§
SEONG HUN JEONG,
YOUNG BAE RYU,
MARCUS J. CURTIS-LONG,
†
†
†
|
HYUNG WON RYU, YOON SU BAEK, JAE EUN KANG, WOO SONG LEE, AND
,
†
KI HUN PARK*
Division of Applied Life Science (BK21 Program), EB-NCRC, Institute of Agriculture & Life
Science, Graduate School of Gyeongsang National University, Jinju 660-701, Korea, 12 New Road,
Nafferton, Driffield, East Yorkshire YO25 4JP, U.K., and Bioindustry Research Center, Korea
Research Institute of Bioscience and Biotechnology, Jeoungeup 580-185, Korea
Twelve polyphenols (1-12) possessing tyrosinase inhibitory properties were isolated from the methanol
(
(
95%) extract of Morus lhou. The isolated compounds consisted of four flavanones (1-4), four flavones
5-8), and four phenylbenzofuranes (9-12). Moracin derivative 12 proved to be new a compound
which was fully characterized. Compounds 1-12 were evaluated for both monophenolase and
diphenolase (the two steps catalyzed by tyrosinase) inhibition to identify the structural characteristics
required for mushroom tyrosinase inhibition. We observed that all parent compounds (1, 5, and 9)
possessing an unsubstituted resorcinol group were highly effective inhibitors of monophenolase activity
(
IC50 values of 1.3, 1.2, and 7.4 µM). The potency of the inhibitors diminished with alkyl substitution
on either the aromatic ring or the hydroxyl functions. Interestingly, flavone 5 was shown to possess
only monophenolase inhibitory activity, but flavanone 1 and phenylbenzofuran 9 inhibited diphenolase
as well as monophenolase significantly. The inhibitory mode of these species was also dependent
upon the skeleton: phenylbenzofuran 9 manifested a simple competitive inhibition mode for
monophenolase and diphenolase; on the other hand flavanone 1 (monophenolase, k
) 0.1966 min-
1
3
-1
-1
app
) 0.0014 min µM-1, k
) 0.0013
) 0.8996 µM) and flavone 5 both showed time-dependent inhibition against
-1
µM , k
4
) 0.0082 min , and K
i
) 0.0468 µM; diphenolase, k
3
4
-
1
app
min , and K
i
monophenolase. Compound 1 operated according to the simple reversible slow binding model whereas
compound 5 operated under the enzyme isomerization model.
KEYWORDS: Tyrosinase; slow-binding; flavonoid; phenylbenzofuran; Morus lhou
INTRODUCTION
dihydroxy phenylalanine (DOPA), a process that is called
monophenolase activity, and the oxidation of DOPA into DOPA
quinine (diphenolase activity), which are the initial steps in the
pathway (4-6). The highly reactive quinines spontaneously
evolve through nonenzymatic coupling to brown pigments of
high molecular weight. Tyrosinase inhibitors usually either
chelate the copper ion within the tyrosinase active site, obstruct-
ing the substrate-enzyme interaction, or prevent oxidation via
an electrochemical process (7-9).
Microorganisms and plants are the main sources of natural
tyrosinase inhibitors. Hydroxylated flavonoids are good target
compounds for tyrosinase inhibitors because they share structural
similarities with L-tyrosine, the natural substrate for tyrosinase
Tyrosinase (EC 1.14.18.1), also known as polyphenoloxidase
PPO), is an essential enzyme in numerous cellular processes
(
including insect molting and the browning of damaged fruit.
Its most well-known trait is the production of melanin for the
protection of skin from UV radiation. However, overproduction
of melanin results in skin hyperpigmentation, characterized by
age spots, melasma and chloasma, all of which are common
maladies (1-3). Thus control of melanin formation is directly
linked to human disease prevention. In addition, the inhibition
of melanin formation is also applicable to fruit preservation by
the alleviation of browning. Tyrosinase is an enzyme containing
binuclear copper. It catalyzes the conversion of tyrosine to 3,4-
(
1). Morus lhou (S.) Koidz. is well renowned as a polyphenol-
rich plant that is one of the most ubiquitous traditional herbal
medicines in East Asia. This species, belonging to the family
of Moraceae, may be considered to be a nontoxic natural
therapeutic agent. Its young leaves and twigs have been
classified as edible by the KFDA (Korea Food & Drug
Administration). Its main bioactive constituents are flavonoids,
*
Corresponding author. Tel: +82-55-751-5472. Fax: +82-55-757-
0
178. E-mail: khpark@gsnu.ac.kr.
†
Gyeongsang National University.
These authors contributed equally to this paper.
‡
§
12 New Road, Nafferton, Driffield, East Yorkshire YO25 4JP, U.K.
|
Korea Research Institute of Bioscience and Biotechnology.
1
0.1021/jf8033286 CCC: $40.75  2009 American Chemical Society
Published on Web 01/23/2009

1196 J. Agric. Food Chem., Vol. 57, No. 4, 2009
Jeong et al.
Figure 1. Chemical structures of isolated compounds 1-12 from the M. lhou.
cumarins, and terpenoids, many of which have been proven to
have antibrowning (10, 11), hypoglycemic (12), antinephritis
(
13), and anti-inflammatory properties (14). Previous workers
reported that this species contains tyrosinase inhibiting fla-
vanones, flavones, stilbenes and phenylbenzofurans. Extracts
of this species, including stilbene derivatives, have been shown
to be effective in the preservation of apple juice against
browning (15). Mulberroside F showed an inhibitory effect on
melanin formation within melanoma cells (16).
As melanin formation is a two-step process from tyrosine,
its formation can be tempered by the inhibition of either stage.
Although some reports have arisen regarding the tyrosinase
inhibitory activities of Moracease extracts, none of these have
been able to disclose the detailed mechanism of enzyme activity
with respect to the phenol. In this study, we isolated twelve
flavonoids from the methanol extract of the roots of M. lhou,
and identified their structures using spectroscopic methods
Figure 2. Selected correlations of HMBC and NOESY for compound 12.
NMR at 500 MHz, 13C NMR at 125 MHz) spectrometer (Bruker,
Karlsruhe, Germany) in CDCl
with TMS as internal standard. EIMS was obtained on a JEOL JMS-
00 mass spectrometer (JEOL, Tokyo, Japan). All reagent grade
3 6 6 4
, acetone-d , DMSO-d , or methanol-d
(
Figure 1).
7
chemicals were purchased from Sigma Chemical Co. (St. Louis,
MO).
MATERIALS AND METHODS
Plant Material. The roots of Morus lhou (S.) Koidz. (No-sang) were
collected at Mt. Bi-Bong in Jinju, Korea, on April, 2006. This plant
was identified by Gyeongsangnam-do Agricultural Research & Exten-
sion Services in Korea.
Extraction and Isolation. The roots of M. lhou(8 kg) were air-
dried, chopped and extracted three times with methanol (15 L × 3,
95%) for 10 days at room temperature. The combined methanol extract
was concentrated in vacuo to yield a dark brown gum (270 g). Vacuum
liquid chromatography (VLC) of the methanol extract on celite was
General Apparatus and Chemicals. Thin-layer chromatography
(
TLC) was carried out using commercially available glass plates
precoated with silica gel (E. Merck Co., Darmstadt, Germany), and
visualized under UV at 254 nm or stained with 10% H SO . Column
chromatography was carried out using 230-400 mesh silica gel
Kieselgel 60, Merck, Germany). Melting points (mp) were measured
on a Thomas Scientific capillary melting point apparatus (Electrothermal
300, U.K.) and are uncorrected. UV spectra were measured on a
Beckman DU650 spectrophotometer (Beckman Coulter, Fullerton, CA).
performed using an increasing gradient of CHCl
CHCl
3
and MeOH. The
fraction (60 g) was chromatographed on silica gel (4 × 60 cm,
3
2
4
230-400 mesh, 400 g) using hexane/EtOAc [40:1 (0.4 L), 30:1 (0.4
L), 20:1 (0.4 L), 10:1 (0.4 L), 5:1 (0.4 L), 1:1 (1 L)] mixtures to furnish
fractions A-E. Fraction D (15 g) was subjected to silica gel chroma-
tography (3 × 60 cm) eluting with hexane/EtOAc (40:1 f 1:2) to give
five fractions (fr. D.1-D.5); fr. D.1-D.2 were resubjected to silica
gel chromatography with hexane/acetone (20:1 f 1:1) followed by
column chromatography on Sephadex LH-20 [eluent: MeOH (2 × 90
(
9
1
13
1
H and C NMR data were all obtained on a Bruker AM 500 ( H

Tyrosinase Slow-Binding Inhibitors of Polyphenols from M. lhou
Table 1. Tyrosinase inhibitory activity of isolated compounds (1-12)
J. Agric. Food Chem., Vol. 57, No. 4, 2009 1197
H-6), 5.90 (1H, J ) 2.0 Hz, H-8), 5.62 (1H, dd, J ) 12.6, 3.0 Hz,
H-2), 5.31 (1H, m, H-2′′), 5.12 (1H, m, H-7′′), 3.23 (2H, d, J ) 7.3
Hz, H-1′′), 3.07 (1H, dd, J ) 17.2, 12.6 Hz, H-3a), 2.72 (1H, dd, J )
17.1, 3.1 Hz, H-3b), 2.11 (2H, m, H-5′′), 2.04 (2H, m, H-6′′), 1.69
L-tyrosine
type of
inhibition (K, µM)
competitive
0.7 ( 0.1)
competitive
L-DOPA
type of
inhibition (K, µM)
IC50
a
b
13
compound IC50 (µM)
i
(µM)
i
(3H, s, H-4′′), 1.63 (3H, s, H-9′′), 1.58 (3H, s, H-10′′); C NMR (125
MHz, methanol-d ) δ 16.6 (C-4′′), 18.2 (C-10′′), 26.3 (C-9′′), 28.2 (C-
4
1
1.3 ( 0.3
26.5 ( 2.2
competitive
(14.1 ( 1.7)
NT
5
′′), 28.9 (C-1′′), 41.3 (C-6′′), 43.6 (C-3), 76.5 (C-2), 96.6 (C-8), 97.4
(
c
(C-6), 103.8 (C-3′), 103.8 (C-4a), 117.8 (C-1′), 121.1 (C-5′), 124.8
(C-2′′), 125.9 (C-7′′), 129.1 (C-6′), 132.6 (C-8′′), 136.9 (C-3′′), 154.9
(C-2′), 157.4 (C-4′), 165.8 (C-5), 165.9 (C-8a), 168.7 (C-7), 198.9 (C-4).
2
47.5 ( 4.0
>200
(
28.3 ( 1.5)
3
4
>200
44.2 ( 0.9
NT
competitive
>200
>200
NT
NT
Kuwanon U (3): amorphous yellow powder; mp 136-137 °C; [R]
D
(
29.7 ( 1.0)
competitive
0.61)
+
-
4
2.4 (c 0.17, CH
3
OH); EIMS m/z (relative intensity) 438 (M , 18%),
5
1.2 ( 0.4
>200
NT
1
20 (29%), 315 (52%), 189 (49%), 153 (100%); H NMR (500 MHz,
CDCl ) δ 6.86 (1H, s, H-6′), 6.37 (1H, s, H-3′), 5.94 (1H, s, H-6),
.93 (1H, s, H-8), 5.51 (1H, dd, J ) 12.8, 2.9 Hz, H-2), 5.18 (1H, m,
H-2′′), 5.03 (1H, m, H-7′′), 3.73 (3H, s, OCH ), 3.19 (2H, m, H-1′′),
2.77-3.19 (2H, H-3), 2.02 (2H, m, H-5′′), 1.974 (2H, m, H-6′′), 1.60
(
3
6
7
>200
127.4 ( 3.9
NT
competitive
>200
>200
NT
NT
5
(
78.3 ( 2.1)
3
8
9
131.8 ( 10.6
7.4 ( 1.0
nt
competitive
>200
64.6 ( 2.2
NT
1
3
competitive
(39.0 ( 0.5)
NT
(3H, s, H-4′′), 1.58 (3H, s, H-9′′), 1.51 (3H, s, H-10′′); C NMR (125
MHz, CDCl ) δ 16.4 (C-4′′), 18.1 (C-10′′), 26.1 (C-9′′), 27.2 (C-5′′),
8.0 (C-1′′), 40.2 (C-6′′), 42.2 (C-3), 55.9 (C4′-OCH ), 78.3 (C-2),
6.0 (C-8), 97.6 (C-6), 100.6 (C-3′), 103.7 (C-4a), 115.1 (C-1′), 122.6
(
4.4 ( 1.6)
3
1
0
160.3 ( 6.7
competitive
>200
2
9
(
(
(
3
(
88.7 ( 3.9)
1
1
1
2
98.5 ( 0.7
82.5 ( 1.2
NT
competitive
>200
>200
NT
NT
C-5′), 123.1 (C-2′′), 124.7 (C-7′′), 127.9 (C-6′), 131.8 (C-8′′), 136.8
C-3′′), 153.7 (C-2′), 159.1 (C-4′), 162.9 (C-5), 164.8 (C-8a), 164.9
C-7), 196.6 (C-4).
(
33.4 ( 2.6)
kojic acid 16.3
NT
NT
NT
8
-Isoprenyl-5′-geranyl-5,7,2′,4′-tetrahydroxy flaVanone (4): amor-
phous yellow powder; mp 161-165 °C; [R] -6.24 (c 0.158, CH OH);
) δ 7.03 (1H, s, H-6′), 6.25 (1H, s,
H-3′), 5.82 (1H, s, H-6), 5.47 (1H, dd, J ) 12.5, 3.0 Hz, H-2), 5.20
a
All compounds were examined in a set of experiments repeated three times;
IC50 values of compounds represent the concentration that caused 50% enzyme
D
3
1
H NMR (500 MHz, methanol-d
4
b
c
activity loss. Values of inhibition constant. Not tested.
(
1H, br t, H-2′′), 5.08 (1H, m, H-10), 4.99 (1H, m, H-7′′), 3.11 (2H,
cm)] to yield compound 4 (8 mg) and subsequently compound 8 (28
mg). Fr. E (18 g) was applied to a silica gel column (3 × 60 cm,
m, H-9), 3.08 (2H, m, H-1′′), 2.65-2.84 (2H, m, H-3), 1.97 (2H, m,
H-6′′), 1.92 (2H, m, H-5′′), 1.57 (3H, s, H-4′′), 1.51 (3H, s, H-12),
2
30-400 mesh, 170 g) [eluent: hexane/acetone (20:1 f 1:1)] to afford
13
1
.50 (6H, s, H-9′′, H-10′′), 1.45 (3H, s, H-13); C NMR (125 MHz,
four subfractions fr. E.1-E.4. Fr. E.2-E.3 were resubjected to silica
gel column {(4 × 70 cm, 230-400 mesh, 330 g) [eluent:hexane/EtOAc
methanol-d
4
) δ 16.7 (C-4′′), 18.2 (C-9′′), 18.5 (C-10′′), 23.0 (C-9), 26.3
(
C-12), 26.5 (C-13), 28.2 (C-6′′), 28.9 (C-1′′), 41.2 (C-5′′), 43.7 (C-3),
6.4 (C-2), 96.7 (C-6), 103.8 (C-4a), 103.9 (C-3′), 109.5 (C-8), 118.3
(
60:1 f 1:2)]} to yield compounds 2 (135 mg) and 3(54 mg). Further
7
elution with hexane/Et
Mixture phase (110 g) was chromatographed on silica gel {(6 × 60
cm, 230-400 mesh, 800 g, eluent: CHCl /acetone [40:1 (1 L), 30:1 (1
L), 20:1 (1 L), 10:1 (1 L), 5:1 (1 L), 1:1 (3 L)]; followed by CHCl
MeOH [20:1 (1 L), 10:1 (1 L), 5:1 (1 L), 1:1 (3 L)]} to give fraction
A-G. Fraction C (6 g) was subjected to silica gel column chromatog-
raphy [eluent: CHCl /acetone (20:1 f 1:1)] followed by Sephadex LH-
3
0 [eluent: MeOH (2 × 90 cm)] yielding 7 (18 mg). Fr. D (15 g) was
applied to a silica gel column [3 × 60 cm, 230-400 mesh, 170 g,
eluent: CHCl /MeOH (90:1 f 4:1)] to afford seven subfractions fr.
D.1-D.7. Fr. D.3-D.5 were subjected to silica gel column [4 × 70
cm, 230-400 mesh, 330 g, eluent: CHCl /MeOH (60:1 f 2:1)] and
then rechromatographed using octadecyl-functionalized silica gel (elu-
ent: MeOH/H O 4/1) to yields compounds 1 (56 mg), 5 (17 mg) and
1 (25 mg). Fr. F (12 g) was chromatographed on silica gel [eluent:
2
O (30:1 f 1:4) generated compound 6 (12 mg).
(
(
(
C-1′), 120.9 (C-5′), 124.4 C-10), 124.9 (C-2′′), 125.9 (C-7′′), 128.9
C-6′), 131.9 (C-11), 132.5 (C-8′′), 136.8 (C-3′′), 154.6 (C-2′), 157.1
C-4′), 162.5 (C-8a), 163.5 (C-5), 166.4 (C-7), 199.3 (C-4).
3
3
/
Norartocarpetin (5): amorphous yellow powder; mp 330-340 °C;
1
H NMR (500 MHz, DMOS-d ) δ 7.76 (1H, d, J ) 8.8 Hz, H-6′), 6.99
6
(
(
(
(
1H, s, H-3), 6.49 (1H, d, J ) 2.3 Hz, H-3′), 6.44 (1H, m, H-5′), 6.43
13
1H, d, J ) 2.1 Hz, H-8), 6.17 (1H, d, J ) 2.1 Hz, H-6); C NMR
125 MHz, DMOS-d ) δ 93.7 (C-8), 98.5 (C-6), 103.1 (C-4a), 103.4
2
6
C-3′), 106.7 (C-3), 107.9 (C-5′), 108.5 (C-1′), 129.7 (C-6′), 157.2 (C-
3
8
1
a), 158.7 (C-2′), 161.3 (C-5), 161.7 (C-2), 161.7 (C-4′), 163.9 (C-7),
81.7 (C-4).
3
1
Morusinol (6): amorphous yellow powder; mp 213-214 °C H NMR
500 MHz, methanol-d ) δ 7.04 (1H, d, J ) 8.9 Hz, H-6′), 6.50 (1H,
4
dd, J ) 10.0, 0.5 Hz, H-9), 6.33 (2H, m, H-5′, H-3′), 5.49 (1H, d, J )
0.0 Hz, H-10), 2.36 (2H, m, H-1′′), 1.50 (2H, m, H-2′′), 1.34 (6H, s,
H-12, H-13), 0.98 (6H, s, H-4′′, H-5′′); C NMR (125 MHz, methanol-
) δ 16.7 (C-4′′), 18.2 (C-9′′), 18.5 (C-10′′), 23.0 (C-9), 26.3 (C-12),
6.5 (C-13), 28.2 (C-6′′), 28.9 (C-1′′), 41.2 (C-5′′), 43.7 (C-3), 76.4
C-2), 96.7 (C-6), 103.8 (C-4a), 103.9 (C-3′), 109.5 (C-8), 118.3 (C-
(
2
1
1
3
CHCl /MeOH (20:1 f 1:1)] to yield compound 9 (34 mg) and
13
Sephadex LH-20 [eluent: MeOH (2 × 90 cm)] to give 10 (4 mg). Fr.
d
4
G and MeOH phase were repeatedly chromatogaphed over silica gel
2
(
1
6
4
3
using [eluent: CHCl /MeOH (20:1 f 1:1)] and then on Sephadex LH-
2
0 [eluent: MeOH (2 × 90 cm)] to yield a new natural product, 12 (77
′), 120.9 (C-5′), 124.4 C-10), 124.9 (C-2′′), 125.9 (C-7′′), 128.9 (C-
′), 131.9 (C-11), 132.5 (C-8′′), 136.8 (C-3′′), 154.6 (C-2′), 157.1 (C-
′), 162.5 (C-8a), 163.5 (C-5), 166.4 (C-7), 199.3 (C-4).
mg). All of the isolated compounds were identified on the basis of the
following spectroscopic data (10, 17-23).
Steppogenin (1): colorless powder; mp 250-254 °C; [R]
D
-3.5 (c
, 288.0634);
) δ 7.18 (1H, d, J ) 8.4 Hz, H-6′),
.34 (1H, d, J ) 2.3 Hz, H-3′), 6.30 (1H, dd, J ) 8.4, 2.3 Hz, H-5′),
.83 (1H, d, J ) 2.0 Hz, H-6), 5.82 (1H, d, J ) 2.1 Hz, H-8), 5.57
Neocyclomorusin (7): amorphous yellow powder; mp 261-264 °C;
0
.125, CH
3
OH); HREIMS m/z 288.0642 (calcd for C15
12 6
H O
1
1
[R] -0.55 (c 0.103, CH OH); H NMR (500 MHz, CDCl ) δ 7.78
H NMR (500 MHz, acetone-d
6
D
3
3
(
1H, d, J ) 8.8 Hz, H-6′), 6.68 (1H, d, J ) 10.0 Hz, H-9), 6.65 (1H,
6
5
dd, J ) 8.8, 2.1 Hz, H-3′), 6.52 (1H, d, J ) 2.1 Hz, H-5′), 6.19 (1H,
s, H-6), 5.52 (1H, d, J ) 10.0 Hz, H-10), 3.96 (1H, dd, J ) 9.2, 2.3
Hz, H-2′′), 3.22 (1H, dd, J ) 16.6, 2.5 Hz, H-1′′a), 2.61 (1H, dd, J )
16.6, 9.3 Hz, H-1′′b), 1.40 (6H, s, H-12, H-13), 1.30 (3H, s, H-4′′),
(
1H, dd, J ) 13.1, 2.9 Hz, H-2), 3.04 (1H, dd, J ) 17.1, 13.1 Hz,
13
H-3a), 2.69 (1H, dd, J ) 17.1, 3.0 Hz, H-3b); C NMR (125 MHz,
acetone-d ) δ 43.1 (C-3), 75.8 (C-2), 96.2 (C-8), 97.2 (C-6), 103.9 (C-
′, 4a), 105.3 (C-5′), 117.9 (C-1′), 129.4 (C-6′), 156.7 (C-2′), 159.9
C-4′), 165.3 (C-8a), 166.1 (C-5), 167.8 (C-7), 198.1 (C-4).
Kuwanon E (2): yellow powder; mp 121 °C; [R] -0.25 (c 0.291,
OH); EIMS m/z (relative intensity) 424 (M , 22%), 406 (52%),
6
1
3
3
(
1.29 (3H, s, H-5′′); C NMR (125 MHz, CDCl ) δ 25.07 (C-4′), 25.4
3
(C-1′′), 25.9 (C-5′′), 28.5 (C-12, C-13), 73.1 (C-3 µ), 78.4 (C-11), 90.9
(C-2 µ), 100.2 (C-6), 101.4 (C-8), 104.4 (C-4a), 108.3 (C-3′), 112.0
(C-5′), 114.7 (C-3), 115.2 (C-9), 116.6 (C-1′), 127.7 (C-10), 130.2 (C-
6′), 152.1 (C-8a), 158.7 (C-2), 159.8 (C-7), 160.2 (C-2‘), 161.5 (C-5),
161.8 (C4′), 181.5 (C-4).
D
+
CH
3
1
3
39 (19%), 301 (32%), 153 (100%); H NMR (500 MHz, methanol-
) δ 7.09 (1H, s, H-6′), 6.37 (1H, s, H-3′), 5.92 (1H, J ) 2.0 Hz,
d
4

1198 J. Agric. Food Chem., Vol. 57, No. 4, 2009
Jeong et al.
Figure 3. (A) Effect of compounds (1, 4, 9, and 12) on the activity of tyrosinase for the catalysis of L-tyrosine. (B) Effect of compounds 1 and 9 on the
activity of tyrosinase for the catalysis of L-DOPA at 30 °C. (C) Relationship between the catalytic activity of tyrosinase and concentrations of compound
1. Concentrations of compound 1 for lines from top to bottom were 0, 0.3, 0.6, 1.0, and 2.0 µM, respectively. (D) Inhibition as a function of preincubation
time for isolated compounds (1, 4, 8, and 9).
1
Kuwanon A (8): amorphous yellow powder; mp 176-177 °C; H
NMR (500 MHz, acetone-d ) δ 6.97 (1H, d, J ) 8.4 Hz, H-6′), 6.64
29.8 (C-13), 40.9 (C-12), 103.0 (C-3), 103.9 (C-4′), 104.3 (C-7, C-6′),
112.6 (C-4a), 113.6 (C-5), 119.3 (C-4), 123.0 (C-2′), 123.5 (C-9), 125.5
(C-14), 132.1 (C-15), 134.1 (C-1′), 136.1 (C-10), 153.9 (C-6), 155.8
(C-7a), 155.9 (C-2), 160.2 (C-3′, C-5′).
6
(
1H, d, J ) 10.0 Hz, H-7′), 6.34 (1H, d, J ) 8.4 Hz, H-5′), 6.19 (1H,
d, J ) 2.1 Hz, H-8), 5.61 (1H, d, J ) 10.0 Hz, H-8′), 4.97 (1H, m,
H-2′′), 2.96 (2H, d, J ) 7.1 Hz, H-1′′), 1.43 (3H, s, H-10′), 1.31 (6H,
Moracinoside M (12): amorphous white powder; mp 175-(decomp)
1
3
s, H-12, H-13), 1.26 (3H, s, H-11′); C NMR (125 MHz, acetone-d
6
)
°C; [R]
D
-7.0 (c 0.291, CH
3
OH); HREIMS m/z 458.1576 (calcd for
1
δ 17.5 (C-5′′), 25.2 (C-1′′), 26.5 (C-4′′), 28.2 (C-10′, C-11′), 78.3 (C-
C H O , 458.1577); H NMR (500 MHz, acetone-d ) δ 7.11 (1H, s,
2
4
2
6
9
6
9
1
1
2
′), 94.7 (C-8), 100.6 (C-6), 104.4 (C-4a), 109.8 (C-5′), 112.6 (C-1′),
15.1 (C-3′), 117.7 (C-7′), 122.6 (C-2′′), 130.7 (C-8′), 131.8 (C-6′),
33.8 (C-3′′), 152.5 (C-2′), 157.1 (C-4, C-8a), 160.0 (C-3), 161.8 (C-
), 164.9 (C-5), 165.0 (C-7), 184.6 (C-4).
H-4), 6.90 (1H, s, H-3), 6.90 (1H, s, H-6′), 6.89 (1H, s, H-4′), 6.74
(1H, s, H-7), 6.45 (1H, s, H-2′), 3.69 (1H, dd, J ) 7.8, 5.5 Hz, H-2′′),
2.96 (1H, d, J ) 16.3, 5.3 Hz, H-1′′a), 2.70 (1H, dd, J ) 16.2, 8.1 Hz,
H-1′′b), 1.23 (3H, s, H-4′′), 1.12 (3H, s, H-5′′); xylopyranoside 4.87
1
Moracin M (9): amorphous yellow powder; mp 261-263 °C; H
(1H, d, J ) 6.4 Hz, xyl-H1), 3.82 (1H, dd, J ) 11.3, 4.9 Hz, xyl-H5
ꢀ
),
NMR (500 MHz, methanol-d
4
) δ 7.24 (1H, d, J ) 8.4 Hz, H-4), 6.81
3.51 (1H, br m, xyl-H4), 3.39-3.45 (2H, m, xyl-H2 and xyl-H3), 3.32
1
3
(
6
1H, s, H-4′), 6.80 (1H, d, J ) 1.8 Hz, H-7), 6.66 (2H, s, H-2′, H-6′),
R 6
(1H, t, J ) 10.5 Hz, xyl-H5 ); C NMR (125 MHz, acetone-d
) δ
.63 (1H, dd, J ) 8.4, 2.1 Hz, H-5), 6.14 (1H, t, J ) 4.0, 1.9 Hz,
21.1 (C-4′′), 26.6 (C-5′′), 32.8 (C-1′′), 70.4 (C-2′′), 78.5 (C-3′′), 99.8
(C-7), 102.8 (C-3), 105.3 (C-2′), 105.7 (C-4′), 106.7 (C-6′), 118.4 (C-
5), 122.2 (C-4), 123.9 (C-4a), 133.7 (C-1′), 153.0 (C-6), 155.8 (C-7a),
155.9 (C-2), 160.1 (C-3′), 160.6 (C-5′); xylopyranoside 66.8 (xyl-C5),
71.1 (xyl-C4), 74.8 (xyl-C3), 77.9 (xyl-C2), 102.9 (xyl-C1). Compound
12 named “moracinoside M” proved to be a new phenylbenzofuran.
Our detailed kinetic analysis unveiled that the kinetic modes of these
inhibitors were significantly affected by their parent skeletons.
Tyrosinase Assays. Mushroom tyrosinase (EC 1.14.18.1) (Sigma
Chemical Co.) was used as described (9, 11) previously with some
modifications, using either, L-DOPA (diphenolase) or L-tyrosine
(monophenolase) as substrate. In spectrophotometric experiments,
1
3
4
H-4′); C NMR (125 MHz, methanol-d ) δ 98.9 (C-7), 102.6 (C-3),
1
4
3
03.9 (C-4′), 104.4 (C-2′, C-6′), 113.7 (C-5), 122.4 (C-4), 123.5 (C-
a), 134.2 (C-1′), 156.6 (C-2), 157.3 (C-7a), 157.7 (C-6), 160.4 (C-
′27, C-5′).
1
Moracin N (10): yellowish powder; H NMR (500 MHz, methanol-
4
d ) δ 7.09 (1H, s, H-4), 6.79 (1H, s, H-7), 6.76 (1H, s, H-3), 6.65 (1H,
s, H-2′), 6.64 (1H, s, H-6′), 6.13 (1H, t, J ) 4.3, 2.2 Hz, H-4′), 5.26
(
1
2
1H, t, J ) 2.8, 1.4 Hz, H-9), 3.25 (2H, m, H-8), 1.65 (3H, s, H-11),
.63 (3H, s, H-12); 13C NMR (125 MHz, methanol-d
6.4 (C-12), 29.9 (C-8), 98.3 (C-7), 102.7 (C-3), 103.8 (C-4′), 104.3
4
) δ 18.2 (C-11),
(C-2′, C-6′), 121.8 (C-4), 123.2 (C-4a), 124.8 (C-8), 126.6 (C-6), 133.3
(C-10), 134.4 (C-1′), 155.0 (C-6), 155.9 (C-7a), 156.2 (C-2), 1660.3
(C-3′, C-5′).
i
enzyme activity was initial velocity (V ) monitored by observing
dopachrome formation at 475 nm with a UV-vis spectrophotometer
(Spectro UV-vis double beam; UVD-3500, Labomed, Inc.) at 30 °C.
All samples were first dissolved in EtOH at 10 mM. First, 200 µL of
1
Albafuran A (11): amorphous yellow powder; mp 131-133 °C; H
NMR (500 MHz, acetone-d
6
) δ 7.08 (1H, d, J ) 8.2 Hz, H-4), 6.85
(
6
1H, s, H-3), 6.76 (2H, s, H-7, H-6′), 6.69 (1H, d, J ) 8.3 Hz, H-5),
a 2.7 mM L-tyrosine (K
m
) 180 µM) or 5.4 mM L-DOPA (K ) 360
m
.24 (1H, s, H-4′), 5.33 (1H, m, H-9), 4.91 (1H, m, H-14), 3.53 (2H,
µM) aqueous solution was mixed with 2687 µL of 0.25 M phosphate
buffer (pH 6.8). Then, 100 µL of the sample solution and 13 µL of the
same phosphate buffer solution containing mushroom tyrosinase (144
units) were added in this order to the mixture. Each assay was conducted
d, J ) 7.3 Hz, H-8), 1.95 (2H, m, H-13), 1.86 (2H, m, H-12), 1.77
1
3
(
3H, s, H-11), 1.42 (3H, s, H-16), 1.37 (3H, s, H-17); C NMR (125
MHz, acetone-d ) δ 16.9 (C-11), 18.1 (C-16), 23.8 (C-8), 26.2 (C-17),
6

Tyrosinase Slow-Binding Inhibitors of Polyphenols from M. lhou
J. Agric. Food Chem., Vol. 57, No. 4, 2009 1199
Figure 4. Time course of oxidation of L-tyrosine catalyzed by mushroom tyrosinase in the presence of compounds (1, 2, 9, and kojic acid). (A) Concentrations
of compounds 1, 2, 9, and kojic acid were 3 µM, 33 µM, 12 µM, and 25 µM. (B) Time-dependent inhibition of tyrosinase in the presence of steppogenin
(1). Conditions were as follows: 180 µM L-tyrosine, 80 units of tyrosinase, and concentrations of steppogenin for curves from top to bottom were 0, 0.25,
0.5, 0.75, 1.0, and 1.5 µM. (C) Time course of the inactivation of tyrosinase by steppogenin. The kobs values at each inhibition concentration were
determined by fitting the data to eq 2. (Inset) Dependence of the values for kobs on the concentration of steppogenin by fitting the data to eq 3. (D)
Time-dependent inhibition of tyrosinase in the presence of steppogenin under the L-DOPA substrate (for curves from top to bottom were 0, 20, 40,
60,100, and 200 µM, respectively).
as three separate replicates. The inhibitor concentration leading to 50%
activity loss (IC50) was obtained by fitting experimental data to the
logistic curve by eq 1 (24):
RESULTS AND DISCUSSION
The chloroform-soluble fraction of the methanolic extract of
Morus lhou (S.) Koidz. roots after separation by chromatography
yielded eleven flavonoids (1-11). Compounds 1-11 are known
compounds and were identified by their spectroscopic data as
steppogenin (1), kuwanon E (2), kuwanon U (3), 8-isoprenyl-
activity (%) ) 100[1 ⁄ (1 + ([I] ⁄ IC ))]
(1)
5
0
Time-Dependent Assays and Progress Curves. Time-dependent
assays and progress curves were carried out using 80 units of tyrosinase,
and L-tyrosine and L-DOPA were used as a substrates (respectively
from monophenolase and diphenolase assays) in 0.25 M phosphate
buffer (pH 6.8) at 30 °C. Enzyme activities were measured continuously
for 10 min on a UV spectrophotometer. To determine the kinetic
parameters associated with time dependent inhibition of tyrosinase,
progress curves with 20 data points (30 s intervals) were obtained at
several inhibitor concentrations using fixed substrate concentrations.
The data were analyzed using the a nonlinear regression program [Sigma
Plot (SPCC Inc., Chicago, IL)] to give the individual parameters for
5
′-geranyl-5,7,2′,4′-tetrahydroxy flavanone (4), norartocarpetin
(
5), morusinol (6), neocyclomorusin (7), kuwanon A (8),
moracin M (9), moracin N (10), albafuran A (11). Activity
guided fractionation of the methanolic extract also gave mora-
cinoside M (12) which was purified over octadecyl-function-
alized silica gel. The structural elucidation of new compound
is detailed below.
Compound 12 had the molecular formula C24
26 9
H O and
twelve degrees of unsaturation, as deduced from HREIMS (m/z
each curve; V
i
(initial velocity), V
s
(steady-state velocity), kobs (apparent
to V ), A (absorbance
i
(apparent K ) according to the following
+
4
58.1576 [M ]) data. The UV spectrum resembled this of
first-order rate constant for the transition from V
i
s
1
13
app
2-phenylbenzofuran derivatives (28). The H and C NMR data
including DEPT experiments showed the presence of twenty-
at 475 nm), and K
equations 24-27):
i
3
2
four carbon atoms: two methylens (sp ), six methins (sp ), five
3
V ⁄ V ) exp(-kobst)
(2)
methins (sp ), two methyls, and nine quaternary carbons. The
0
13
C NMR data enabled carbons corresponding to seven aromatic
app
kobs ) k (1 + [I] ⁄ Ki
)
(3)
(4)
C-C double bonds to be identified, thus accounting for seven
of twelve degrees of unsaturation. The remaining five degrees
of unsaturation were ascribed to cyclic systems (including the
benzofuran ring).
4
A ) V t + (V -V )[1 - exp(-kobst)] ⁄ k
obs
s
i
s
A plot of the natural log of the residual enzyme activity versus
The presence of an epoxyprenyl group was deduced from
preincubation time gave a straight line with a slope of -kobs. The values
app
the connectivity between methylene protons H-1′′ (δ 2.96, 2.70)
of k
3
, k
4
, and K
i
were calculated from the plot of the kobs versus
H
concentration of inhibitors according to the method of Morrison and
Walsh (24-26).
and methane proton H-2′′ (δ
H
3.67) in the COSY spectrum and
78.5), 4′′ (δ
correlation of H-2′′ with three carbons, C-3′′ (δ
C
C

1200 J. Agric. Food Chem., Vol. 57, No. 4, 2009
Jeong et al.
Figure 5. (A) Progress curves for the competitive behavior of steppogenin. Conditions were as follows: 1.0 µM steppogenin, 80 units of tyrosinase, and
concentration of L-tyrosine for curves from top to bottom were 0, 900, 700, 360, and 180 µM. (Inset) Dependence of the values for kobs on the concentration
of L-tyrosine. (B) Progress curves for the competitive behavior of steppogenin using L-DOPA as substrate. Conditions were as follows: 100 µM steppogenin,
8
0 units tyrosinase, and concentration of L-DOPA for curves from top to bottom were 0, 900, 700, 360, and 180 µM. (Inset) Dependence of the values
for kobs on the concentration of L-DOPA.
1.0), 5′′ (δ 26.6) in the HMBC spectrum. The two isolated
2
C
that was ascertained by GC/MS analysis with reference to an
authentic sample. The xylopyranosyl moiety was placed at C-3′
of the B-ring by HMBC. Thus, compound 12 was identified as
5′-[2,3-epoxy-3-methylbutyl]-3′-O-ꢀ-D-xylopyranosyl-6,5′-dihy-
droxy-2-phenylbenzyfuran called moracinoside M.
protons of H-4 and H-7 in the A-ring were observed at 7.11
and 6.74 ppm. The H-3 furan proton in the C-ring occurred at
6
Thus, H-2′ and H-4′ appeared at 6.44 ppm and 6.88 ppm as
singlet (absence of meta coupling). The abovementioned ep-
oxyprenyl group was placed at C-5 on the A-ring due to HMBC
correlation of C-5 with H-1′′ and H- 2′′ (Figure 2). The H
NMR spectrum exhibited a doublet at δ 4.87 (J ) 6.4 Hz)
indicating the presence of an anomeric carbon which constituted
the starting point of COSY analysis. The assignment of pyran
structure was determined on the basis of successive connec-
tivities from xyl-1 to xyl-5. In 2D-NOESY experiments in the
xylose, an NOE cross peak was observed between syn-1,3-
.90 ppm with same chemical shift value with H-6′ in the B-ring.
As shown in Table 1, all polyphenols investigated apart from
compounds 3 and 6 exhibited a significant (dose-dependent)
degree of monophenolase (L-tyrosine substrate) inhibition [(IC50
1
H
1
.2-160.3 µM) (Figure 3A)]. As the concentrations of the
inhibitors increased, the residual enzyme activity rapidly
diminished. Compounds 1, 2, 4, 5, 9, 11, and 12 emerged to be
the most potent inhibitors to monophenolase activity, with IC50
s
values 1.3, 47.5, 44.2, 1.2, 7.4, 98.5, and 82.5 µM, respectively.
It seems that alkyl substitution on any position of the aromatic
rings within the parent compounds (1, 5, and 9) reduces the
potency of the inhibitors greatly. The markedly low potency of
compound 3 relative to compound 1 suggests that free hydroxyl
functions are also paramount to inhibitor activity. As we were
interested in each step of the oxidation process, we proceeded
to investigate the inhibition of diphenolase activity by these
compounds. Interestingly, only two of these compounds, fla-
vanone (1) and phenylbenzofuran (9), displayed significant
inhibitory activities against diphenolse with IC50s of 26.5 and
64.6 µM, respectively (Figure 3B). It is noteworthy that these
diaxial protons xyl-H5
xyl-H4 only showed a cross peak with xyl-H5
we anticipated, there are no NOE cross peaks between xyl-1,
xyl-2, xyl-3 and xyl-4, which all have a trans diaxial relationship
between each other. The cis relationship between xyl-H4 and
R
and xyl-H1 (anomeric proton), whereas
(Figure 2). As
ꢀ
xyl-H5
corresponding to xyl-H5
Hz (vicinal)]. The relative stereochemistry of xyl-H1 and xyl-
H2 was easily confirmed by the J value of anomeric proton (δ
.87, J ) 6.4 Hz) that is a characteristic of ꢀ-xylopyranosyl
moiety (29, 30). Acid hydrolysis of 12 yields ꢀ-D-xylose a result
ꢀ
is also confirmed by the J value of the double doublet
ꢀ
[J ) 11.0 Hz (germinal), J ) 5.0
1
2
H
4

Tyrosinase Slow-Binding Inhibitors of Polyphenols from M. lhou
J. Agric. Food Chem., Vol. 57, No. 4, 2009 1201
Figure 6. (A) Dixon plots for the inhibition of compound 1 on the monophenolase activity of tyrosinase. (Inset) Replot of slope versus the corresponding
/[S] of compound 1. In the presence of different concentrations of substrate for lines from bottom to top: 360, 180, and 90 µM. (B) and (D) Lineweaver-Burk
plots for the inhibition of compounds 1 and 9 on the diphenolase activity of tyrosinase. (C) and (E) Lineweaver-Burk plots for the inhibition of compounds
and 12 on the monophenolase activity of tyrosinase. Conditions were as follows: 180 µM L-tyrosine, 360 µM L-DOPA, 144 units of tyrosinase, 0.25
M phosphate buffer (pH 6.8), at 30 °C. In the presence of different concentrations of compounds for lines from bottom to top: (B) for compound 1, 0,
1
9
1
0, 25, and 40 µM; (C) for compound 9, 0, 3.7, 7.4, and 14.8 µM; (D) for compound 9, 0, 30, 60, and 120 µM; (E) for compound 12, 0, 40, 80, and
60 µM.
1
values are more than 20-fold greater than the corresponding
IC50s for monophenolase.
The inhibition mechanisms displayed by the isolated polyphe-
nols were subsequently studied. All inhibitors manifested the
same relationship between enzyme activity and enzyme con-
centration. The inhibition of mushroom tyrosinase by compound
of L-tyrosine catalyzed by tyrosinase in the presence of
compounds 1, 2, 9, and kojic acid. As expected, in the presence
of monophenolase inhibitors, the lag time was prolonged from
60 s in the control to 210 s with addition of inhibitors. Also,
the lag time of positive control was 210 s at 25 µM. Compound
9 emerged not to be a time-dependent inhibitor whereas the
other inhibitors (1 and 2) showed time-dependent inhibition
profiles (Figure 4A). These results echo the preincubation
experiments (Figure 3D). The potent tyrosinase inhibitor,
flavanone (1), showed a typical progress curve for slow-binding
inhibition at low concentrations. Slow-binding inhibition mech-
anisms can be investigated by preincubation of the enzyme with
inhibitor followed by measurement of initial velocities for
substrate oxidation as a function of preincubation time. Increas-
ing flavanone (1) concentration led to a decrease in both the
1
is illustrated in Figure 3C representatively. Plots of the initial
velocity versus enzyme concentration in the presence of different
concentrations of steppogenin (1) gave a family of straight lines,
all of which passed through the origin. Increasing the inhibitor
concentration resulted in a lowering of the line gradients,
indicating that these compounds were reversible inhibitors. To
investigate the enzyme activity as a function of time exposed
to the inhibitor, we measured residual enzyme activity of
preincubated enzyme with inhibitors over a number of exposure
times. Flavanones (1 and 4) and flavone (8) showed a time-
dependent inhibitory effect on tyrosinase activity, whereas
phenylbenzofuran (9) did not. In our previous work, it was
proven that flavone (5) showed a time-dependent inhibitory
effect (11).
initial velocity (V
The progress curves obtained using various concentrations
of the inhibitors were fitted to eq 2 to determine V , V
i
s
) and the steady-state rate (V ) (Figure 4B).
i
s
, and kobs.
The kobs values were plotted as a function of steppogenin
concentration. The results indicated that steppogenin inhibits
mushroom tyrosinase by simple reversible slow binding when
L-tyrosine was used as a substrate (Figure 4C). This was backed
We then undertook the full kinetic characterization of these
inhibitors. Figure 4A depicts the time course for the oxidation

1202 J. Agric. Food Chem., Vol. 57, No. 4, 2009
Jeong et al.
up by the observation that the kobs values exhibited a linear
dependence on the inhibitor concentration as shown in Figure
In conclusion, twelve polyphenols were successfully isolated
and purified from M. lhou including a new phenylbenzofuran
(12), which we named “moracinoside M”. The inhibitory
potencies and capacities of these flavonoids toward the monophe-
nolase activity of mushroom tyrosinase were studied in detail.
Interestingly, they showed different inhibitory modes depending
upon the skeleton: simple reversible slow-binding (time de-
pendent) for flavanones, enzyme isomerization for flavones, and
a simple competitive inhibition for phenylbenzofurans (time
independent). The enzymatic oxidation of tyrosine to melanin
is of considerable importance since melanin has many important
functions and alterations in melanin synthesis occur in many
diseases. The flavonoid-derived tyrosine inhibitors studied herein
displayed the most potent inhibition profiles suggesting that they
are excellent candidates to be developed as effective antibrown-
ing agents for foodstuff of skin whitening agents in cosmetics.
4
C inset. Also, Figure 4D showed typical progress curves for
slow binding behavior, when the oxidation of L-DOPA was
catalyzed by mushroom tyrosinase in the presence of different
steppogenin concentrations. Thus, analysis of data according
to eqs 2 and 3 yielded the following values: k
3
) 0.196605
) 0.04684 µM.
Steppogenin also emerged as a simple reversible slow-binding
-1
-1
-1
app
min µM , k
4
) 0.009209 min , K
i
) 0.001445 min- µM , k
1
-1
inhibitor of diphenolase (k
3
4
)
-1
app
0
.0013 min , K
i
) 0.8996 µM). Similar kinetic analysis
previously reported from our group delineated that flavone 5
effected slow-binding enzyme isomerization. Thus, despite
having similar molecular size and functionality, and almost equal
inhibitory potencies toward monophenolase, steppogenin inhibits
both monophenolase and diphenolase via the simple reversible
slow-binding mode whereas flavone (5) was active only against
monophenolase through enzyme isomerization (11).
ABBREVIATIONS USED
Most slow-binding enzyme inhibitors act as competitive
inhibitors, binding at the enzyme active site (24, 26), although
it is possible for them to interact with the enzyme by competi-
tive, noncompetitive, or uncompetitive inhibition patterns. To
distinguish the mode of inhibition of a time-dependent inhibitor,
it is convenient to analyze the effect of various substrate
concentrations of kobs at fixed inhibitor concentration. A
competitive inhibitor will display a decrease of kobs with
increasing substrate concentrations, while for a noncompetitive
inhibitor the value of kobs is independent of substrate concentra-
tions. Figure 5A illustrates typical progress curves of time-
dependent inhibition of steppogenin (1.0 µM) when the enzy-
matic reaction is initiated by the addition of tyrosinase (80 units),
in the presence of various concentrations of L-tyrosine (180,
IC50, the inhibitor concentration leading to 50% activity loss;
app
K
i
, inhibition constant; K
maximum velocity; K
first-order rate constant for the transition from V
velocity; V steady-state rate; A, absorbance at 475 nm.
i
i
, apparent K; k, rate constant; Vmax,
m
, Michaelis-Menten constant; kobs, apparent
to V ; V, initial
i
s
i
s
LITERATURE CITED
(
1) Fu, B.; Li, H.; Wang, X.; Lee, F. S. C.; Cui, S. Isolation and
identification of flavonoids in licorice and a study of their
inhibitory effects on tyrosinase. J. Agric. Food Chem. 2005, 53,
7
408–7414.
(
2) Meada, K.; Fukuda, M. J. In vitro effectiveness of several
whitening cosmetic components in human melanocytes. Soc.
Cosmet. Chem. 1991, 42, 361–368.
3
60, 700, and 900 µM). The kobs values were determined by
fitting data to eq 4. As a result, steppogenin is a competitive
inhibitor because kobs decreased with increasing substrate
concentration (inset). When L-DOPA was used as a substrate,
steppogenin also exhibited competitive inhibition (Figure 5B).
Recently, many researchers reported that resorcinol can be
classified as slow-binding competitive inhibitor of mushroom
tyrosinase. Consistent with the structural similarities to the
inhibitors employed in the above results, our inhibitors also show
time dependent inhibition behavior.
(3) Mcevily, J. A.; Iyengar, R.; Otwell, Q. S. Inhibition of enzymatic
browning in food and beverages. Crit. ReV. Food Sci. Nutr. 1992,
3
2, 253–273.
(
4) Halaouli, S.; Asther, M.; Sigoillot, J. C.; Hamdi, M.; Lomascolo,
A. Fungal tyrosinases: New prospects in molecular characteristics,
bioengineering and biotechnological applications. J. Appl. Mi-
crobiol. 2006, 100, 219–232.
(5) Mayer, A. M. Polyphenol oxidases in plants and fungi: Going
places? A review. Phytochemistry 2006, 67, 2318–2331.
(6) Fenoll, L. G.; Penalver, M. J.; Rodriguez-Lopez, J. N.; Varon,
R.; Garcia-Canovas, F.; Tudela, J. Tyrosinase kinetics: Discrimi-
nation between two models to explain the oxidation mechanism
of monophenolase and diphenolase substrates. Int. J. Biochem.
Cell Biol. 2004, 36, 235–246.
7) Kubo, I.; Nihei, K. I.; Shimizu, K. Oxidation products of quercetin
catalyzed by mushroom tyrosinase. Bioorg. Med. Chem. 2004,
12, 5343–5347.
8) Matsuura, R.; Ukeda, H.; Sawamura, M. Tyrosinase inhibitory
activity of citrus essential oils. J. Agric. Food Chem. 2006, 54,
Finally, we investigated the characteristics of inhibitors 1, 9,
and 12 with respect to the two different steps carried out by the
enzyme individually. In this experiment, the initial velocity of
the enzyme was monitored by observing dopachrome formation
at 475 nm. Compound 1 displayed competitive inhibition against
(
both monophenolase (K
i
) 0.7 µM) and diphenoalse (K ) 14.1
i
(
µM) as shown in Dixon (Figure 6A) and Lineweaver-Burk
plots (Figure 6B) respectively. Phenylbenzofuran (9) showed
2
309–2313.
competitive inhibition against both monophenolase (K
µM) and diphenolase (K ) 64.6 µM) by Lineweaver-Burk
plots. The new compound 12 also showed competitive inhibition
(K ) 33.4 µM) Lineweaver-Burk plot]. Most competitive
i
) 4.4
(
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[
i
inhibitors of tyrosinase bind to the active site mimicking the
enzyme/substrate interaction. The B-ring of our isolated com-
pounds is very similar to tyrosinase substrates (tyrosine and
DOPA). This leads to the competitive displacement of substrates
from the active site of the cofactor in a lock-and-key model.
Some flavonoids have been described to chelate copper (7)
which has been suggested as a mode of inhibition. However,
(
2+
no shift in the UV-visible spectra was observed by adding Cu
to these species.

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Received for review August 27, 2008. Revised manuscript received
December 14, 2008. Accepted December 14, 2008. This study was
supported by a grant of the Korea Healthcare technology R&D Project,
Ministry of Health & Welfare (A080813), the MOST/KOSEF to the
Environmental Biotechnology National Core Research Center (R15-
(
(
22) Purusotam, B.; Shigetoshi, K.; Satoshi, T.; Mineo, S.; Tsuneo,
N. Two new 2-arylbenzofuran derivatives from hypoglycemic
activity-bearing fractions of Morus insigmis. Chem. Pharm. Bull.
2
2
003-012-0200-0), and the MEST/KOSEF (Nuclear R&D Program,
007-00614), Republic of Korea.
1
993, 41, 1238–1243.
JF8033286