Inhibition of tyrosinase by flavonoids, stilbenes and related 4- substituted resorcinols: Structure-activity investigations

Article abstract of DOI:10.1055/s-2000-11113

Several flavonoids, stilbenes and related 4-substituted resorcinols, obtained from Artocarpus incisus and other plants or synthesized, were tested for their inhibitory activity against tyrosinase. The structure-activity relationships suggested that specific natural or synthesized compounds having the 4-substituted resorcinol skeleton have potent tyrosinase inhibitory ability. Kinetic studies have indicated that specific compounds having the 4- substituted resorcinol skeleton exhibit competitive inhibition of the oxidation of DL-β-(3,4-dihydroxyphenyl)alanine (DL-DOPA) by mushroom tyrosinase. These findings could lead to the design and discovery of new tyrosinase inhibitors.

Full text of DOI:10.1055/s-2000-11113

Original Paper
11
Inhibition of Tyrosinase by Flavonoids, Stilbenes and Related
4-Substituted Resorcinols: Structure-Activity Investigations
Kuniyoshi Shimizu, Ryuichiro Kondo*, and Kokki Sakai
Department of Forest Products, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Received: March 18, 1999; Accepted: June 27, 1999
Revision accepted: June 27, 1999; Received: March 18, 1999
Abstract: Several flavonoids, stilbenes and related 4-substitut-
the inhibitory activity of flavonoids, stilbenes and their conge-
ners, including some related 4-substituted resorcinols.
ed resorcinols, obtained from Artocarpus incisus and other
plants or synthesized, were tested for their inhibitory activity
against tyrosinase. The structure-activity relationships suggest-
ed that specific natural or synthesized compounds having the
4-substituted resorcinol skeleton have potent tyrosinase inhibi-
tory ability. Kinetic studies have indicated that specific com-
pounds having the 4-substituted resorcinol skeleton exhibit
competitive inhibition of the oxidation of DL-b-(3,4-dihydroxy-
phenyl)alanine (DL-DOPA) by mushroom tyrosinase. These find-
ings could lead to the design and discovery of new tyrosinase
inhibitors.
Materials and Methods
Chemicals
The compounds [(±)-pinocembrin (2) (3), (+)-aromadendrin
(4) (4), ()-fustin (5) (5), ()-taxifolin (6) (6), (+)-dihydromyri-
cetin (2) (7), chrysin (11) (8), kaempferol (13) (9), quercetin
(14) (5), myricetin (15) (10), pinosylvin (21) (11), oxyresvera-
trol (22) (12) and bis(2,4-dihydroxyphenyl)methane (34)
(13)] were provided by the Laboratory of Wood Chemistry,
Department of Forest Products, Faculty of Agriculture, Kyushu
University in Japan, and their purities and identification had
been confirmed by comparison with references. The following
reagents were purchased: [()-flavanone (1), flavone (10), 2,4-
dihydroxybenzaldehyde (26), 2,4-dihydroxy-N-(2-hydroxy-
ethyl)benzamide (29), 2,4-dihydroxybenzophenone (30), 4-
hexylresorcinol (38) and 4-dodecylresorcinol (39) from Al-
drich Chem. Co.], [()-naringenin (3) and morin (17) from Sig-
ma Chem. Co.], [2,4-dihydroxyacetophenone (27), 2,4-dihy-
droxybenzoic acid (28), resorcinol (32), L-tyrosine and DL-b-
(3,4-dihydroxyphenyl)alanine (DL-DOPA) from Wako Pure
Chemical Industries, Ltd.)], [4-(2-pyridylazo)resocrinol (31),
4-(2-thiazolylazo)resorcinol (33) from Dojindo Laboratories]
and [4-chlororesorcinol (35), 4-ethylresorcinol (40) from To-
kyo Kasei Kogyo Co., Ltd.]. The reagents (+)-dihydromorin (8),
(+)-norartocarpanone (9), apigenin (12), artocarpin (16), arto-
carpesin (18), isoartocarpesin (19), 4-prenyloxyresveratrol
(23), chlorophorin (24), artocarbene (25) (2) and (±)-angolen-
sin (20) (14) were isolated previously.
Key words: 4-Substituted resorcinols, tyrosinase inhibitors, Ar-
tocarpus incisus, Moraceae, structure-activity relationship.
Introduction
The color of mammalian skin and hair is determined by a
number of factors, the most important of which is the degree
and distribution of the melanin pigmentation. Melanin bio-
synthesis inhibitory compounds are useful not only as skin-
whitening agents used in cosmetics but also as a remedy for
disturbances in pigmentation. Tyrosinase (phenol oxidase) is
known to be a key enzyme for melanin biosynthesis in plants,
microorganisms and mammalian cells (1). Therefore, many
tyrosinase inhibitors have been tested in cosmetics and phar-
maceuticals as a way of preventing overproduction of melanin
in epidermal layers. Also, tyrosinase is one of the most impor-
tant key enzymes in the insect molting process, and investiga-
tion on its inhibitors may be important in finding alternative
insect control agents. Melanin formation is considered to be
deleterious to the color quality of plant-derived food. This
broadens the possible use of tyrosinase inhibitors as food ad-
ditives, in addition to insect control agents and whitening
agents. These observations led us to search for naturally oc-
curring tyrosinase inhibitors. In our previous screening of the
mushroom tyrosinase inhibitor, seven compounds from the
heartwood extracts of Artocarpus incisus L. f. (Moraceae)
showed potent inhibitory activity (2). This paper deals with
4-Methylresorcinol (36), 4-(phenylmethyl)resorcinol (37) and
4-propylresorcinol (41) were prepared by reduction of 26, 30
and 2¢,4¢-dihydroxypropiophenone (Aldrich Chem. Co.) with
NaBCH3CN (Aldrich Chem. Co.), respectively. EIMS, m/z: 36
(M+: 124), 37 (M+: 200), 41 (M+: 152).
Enzyme assays
Mushroom tyrosinase [EC 1. 14. 18. 1] activity was determined
by using L-tyrosine or DL-DOPA as the substrate. L-Tyrosine oxi-
dation assay was done as described previously (2). DL-DOPA
oxidation assay of 0.1 ml of mushroom tyrosinase solution
(625 U/ml, Wako Pure Chemical Industries, Ltd.): 0.7 ml of DL-
Planta Medica 66 (2000) 11±15
ꢀ Georg Thieme Verlag Stuttgart New York
·
ISSN: 0032-0943

12 Planta Med. 66 (2000)
Kuniyoshi Shimizu, Ryuichiro Kondo, and Kokki Sakai
DOPA buffer solution (2.0 mM), 0.1 ml of McIlvain buffer (pH
6.8) and 0.1 ml of DMSO with or without sample were mixed
and incubated at 25 8C. A control reaction was conducted
without the test sample. The absorbance was measured at
475 nm before and after incubation. The percentage of inhibi-
tion of tyrosinase was calculated as follows: tyrosinase inhibi-
tion (%) = (A ± B)/A ” 100, where A represents the difference
in the absorbance of the control sample between the incuba-
tion time of 0.35 and 0.45 min, and B represents the difference
in the absorbance of the test sample between the incubation
time of 0.35 and 0.45 min. The results were from the three
concurrent readings and each S.D. was usually within 2% of
the mean. Kojic acid (Tokyo Kasei Kogyo Co., Ltd.) was used as
a positive standard.
Results and Discussion
To study structure-activity relationships, several flavonoids
and stilbenes were tested for their inhibitory activity on tyro-
sinase (substrate: L-tyrosine) by measuring the concentration
required to effect a 50% inhibition of enzyme activity (IC50).
Nine compounds (8, 9, 12, 16, 18, 19, 23, 24 and 25) isolated
from A. incisus were previously examined for their inhibitory
activity towards mushroom tyrosinase (2). Seven of these (12
and 16 were not included) exhibited potent tyrosinase inhibi-
tory activity [Table 1, kojic acid (positive standard, substrate:
L-tyrosine): IC50 = 8.66mM (2)]. Interestingly, the active com-
pounds had 4-substituted resorcinol as a common skeleton
(Fig.1). This brief structure-activity relationship could mean
that the 4-substituted resorcinol skeleton is important for re-
vealing the tyrosinase inhibitory activity. In addition, it
should be noted that artocarpin (16) did not show inhibitory
activity, in spite of having a 4-substituted resorcinol skeleton
at ring B. Therefore, to clarify which substructure is important
to reveal the tyrosinase inhibitory effect, further structure-ac-
tivity relationships were examined in detail. The test com-
pounds were flavonoids and stilbenes isolated from various
plants, synthesized or commercially available. The results
were summarized in Table 1 and Fig. 2.
Fig.2 The chemical structures of 1±24.
tory activity, but one (21) did not. These results can be ex-
plained by the fact that hydroxylation of 21, resulting in 22,
increases its inhibitory activity dramatically. Also, the addi-
tion of isoprenyl chain (prenyl or geranyl) to the stilbenes
having a 4-substituted resorcinol skeleton slightly increased
their inhibitory activities (22: IC50 = 0.98mM ® 23: IC50
=
0.66mM ® 24: IC50 = 0.26mM). Resveratrol (3,4¢,5-trihydroxy-
stilbene), 3,5-dihydroxy-4¢-methoxystilbene, 3,4¢-dimethoxy-
5-hydroxystilbene, trimethylresveratrol and piceid (4-O-b-D-
glucosylresveratrol) showed much lower inhibitory effects
than oxyresveratrol (22) on dopa oxidase activity of mush-
room tyrosinase (15). Therefore, in the case of stilbenes, the
4-substituted resorcinol skeleton must be the most important
feature for revealing potent tyrosinase inhibition.
Among five stilbenes (21±25), four (22±25) having a 4-sub-
stituted resorcinol skeleton showed potent tyrosinase inhibi-
Among 20 flavonoids, only 4 flavonoids (8, 9, 18 and 19),
which have the 4-substituted resorcinol skeleton at ring B,
showed potent tyrosinase inhibitory activity. Glabridin (one
of the isoflavans) (16), kurarinone (flavanone), kushenol N (di-
hydroflavonol) and kosamol A (dihydroflavonol) (17) were re-
ported as potent tyrosinase inhibitors that have a common 4-
substituted resorcinol skeleton at ring B. In contrast, 16, 17
and 20 did not show tyrosinase inhibitory activity, in spite of
having a 4-substituted resorcinol skeleton at ring B. These re-
sults indicate that for flavonoids not only a 4-substituted re-
sorcinol skeleton but also additional structural factors are
necessary to reveal tyrosinase inhibitory activity.
In the case of flavonoids having a 4-substituted resorcinol
skeleton, except for 20 (which belongs to the a-methyldeoxy-
benzoins), the flavanone type compounds (flavanones and
their C3 substituted derivatives) were more potent inhibitors
than the flavone type compounds (flavones and their C3 sub-
stituted derivatives), e.g., 8 showed a stronger inhibitory ef-
fect than the corresponding flavone 17. Introduction of a C3
substituent to the flavanone (9 ® 8) and flavone type (18 and
19 ® 16 and 17) dramatically decreased their activity. Thus,
even in flavonoids having a 4-substituted resorcinol skeleton,
Fig.1 The chemical structures and IC50 of active components from
A. incisus. The boxed part: 4-substituted resorcinol skeleton.

Structure-Activity Investigations
Planta Med. 66 (2000) 13
Table 1 Inhibitory activity of flavonoids and stilbenes on tyrosinase (substrate: L-tyrosine).
No Name
R3
R5
R6
R7
R2¢
R3¢
R4¢
R5¢
(C2, C3)
IC50 (mM)
1
()-flavanone
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2S,2R
2S
>200
2
(±)-pinocembrin
()-naringenin
(+)-aromadendrin
()-fustin
H
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
>200
3
H
H
H
OH
OH
OH
OH
OH
OH
OH
H
H
2S,2R
>200
lag time decreasea
4
OH
OH
OH
H
H
H
(2R,3R)
5
H
OH
OH
OH
H
H
(2R,3R),(2S,3S) lag time decreasea
(2R,3R),(2S,3S) lag time decreasea
6
()-taxifolin
OH
OH
OH
OH
H
H
H
7
(+)-dihydromyricetin OH
H
OH
H
(2R,3R)
(2R,3R)
2S
lag time decreasea
25b
1.76b
8
(+)-dihydromorin
(+)-norartocarpanone
flavone
OH
H
OH
OH
H
9
H
H
10
11
12
13
14
15
16
H
H
H
>200
chrysin
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
H
H
>200
>185b
apigenin
OH
OH
OH
OH
H
H
OH
OH
OH
OH
OH
H
kaempferol
quercetin
OH
OH
OH
Pr
H
H
H
103
H
OH
OH
H
H
lag time decreasea
lag time decreasea
>228b
myricetin
H
OH
H
artocarpin
CHCHCH
(CH3)2
OCH3 OH
17
18
19
morin
OH
H
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
OH
OH
OH
H
H
H
>330
13.5b
21.1b
artocarpesin
isoartocarpesin
Pr
H
CHCHCH
(CH3)2
20
(±)-angolensin
>200
R3
R4
H
R5
R2¢
H
R4¢
H
21
22
23
24
25
pinosylvin
OH
OH
OH
OH
OH
OH
>46
oxyresveratrol
H
OH
OH
OH
OH
OH
OH
0.98
0.66b
0.26b
2.45b
4-prenyloxyresveratrol OH
Pr
Ger
chlorophorin
artocarbenec
OH
a means promotion effect which could act as cofactor like diphenol (1).
b Obtained from data of Ref. (2).
c See Fig.1.
introduction of a C3 substituent decreased the inhibitory ac-
tivity, probably because of its steric hindrance.
ilar results were given by 9, 22, 24, 25 and 40 (Table 3). Com-
pounds 8 and 32 did not show typical inhibitory patterns. In-
terestingly, these compounds (8 and 32) exhibited some stim-
ulatory activity to the enzyme at low concentration, similar to
a previous report (18). The results obtained so far suggest that
(a) 8 and the corresponding flavanone 9 not possessing a C3-
hydroxy group affect mushroom tyrosinase in different ways,
and that (b) 32 and the corresponding 4-substituted resorci-
nols (9, 22±25 and 40) affect mushroom tyrosinase in differ-
ent ways. However, further work is needed to clarify the in-
hibitory mechanism of 8 and 32.
Compound 20 did not show tyrosinase inhibitory activity, in
spite of having a 4-substituted resorcinol skeleton. To clarify
which substructure causes inactivity of 20, we examined the
effects of different C4 substituents on the tyrosinase inhibito-
ry activity of the 4-substituted resorcinols (Table 2). Table 2
demonstrates the powerful influence of the C4 substituent on
the potency of these compounds. Surprisingly, introduction of
a carbonyl substituent (26±30) decreased the inhibitory ac-
tivity dramatically. Also, compounds having an azo substitu-
ent (31, 33) showed much lower inhibitory activity than 22±
25, in spite of having a similar shape to stilbenes concerning
the double bond. Introductions of chlorine (35), alkyl (36±41)
or phenylmethyl (34) substituents at C4 resulted in potent in-
hibitory activities. The non-substituted 32 did not show a po-
tent inhibitory activity.
Thus, the C4-substituents of resorcinol derivatives and the
C3-substituents of flavonoids that have a 2¢,4¢-dihydroxy-
phenyl skeleton seem to significantly affect tyrosinase activi-
ty.
The tyrosinase inhibitory effects (IC50, Ki and inhibition type)
of representative 4-substituted resorcinols using DL-DOPA as a
substrate are shown in Table 3. Compound 40 showed a
stronger inhibitory activity than that of kojic acid, using both
L-tyrosine and DL-DOPA as a substrate but the inhibitory ef-
fects of 9 and 22±24 were weaker than that of kojic acid us-
ing DL-DOPA as a substrate, in spite of showing much stronger
Kinetic studies were carried out with the five active com-
pounds (8, 9, 23±25) from A. incisus, as well as the related
compounds (22, 32 and 40). The Lineweaver-Burk plot of 23
for DL-DOPA as a substrate is shown in Fig. 3. The mode of in-
hibition of tyrosinase by 23 was competitive. In addition, sim-

14 Planta Med. 66 (2000)
Kuniyoshi Shimizu, Ryuichiro Kondo, and Kokki Sakai
inhibitory activity using L-tyrosine as a substrate. Thus, the or-
der of inhibitory effects of these compounds having a 4-sub-
stituted resorcinol skeleton were different depending on
whether L-tyrosine or DL-DOPA was used as a substrate, in
comparison with kojic acid.
hibitors of mushroom tyrosinase (19). Therefore, the differ-
ence in the inhibitory type of oxyresveratrol against tyrosi-
nase between us and Shin et al. (15), seems to be due to esti-
mated tyrosinase inhibitory activity by different limited reac-
tion times. To characterize the behavior of these inhibitors
completely, a further kinetic study must be needed in order to
determine the kinetic parameters (KI, K¢I and k6) according to
(19). However, in this study, the results of IC50 by using assays
with limited reaction time are a worthy and valid parameter
for understanding the structure-activity relationships.
Oxyresveratrol (22) showed a competitive inhibitory type ac-
tivity in this study, although it was recently reported as a non-
competitive inhibitor on mushroom tyrosinase with L-DOPA
as a substrate (15). The difference may be explained as fol-
lows. It was reported recently that 4-substituted resorcinols
such as 4-ethylresorcinol, 4-hexylresorcinol and 4-dodecylre-
sorcinol could be classified as slow-binding competitive in-
Some 4-substituted resorcinols have been reported as inhibi-
tors of enzymatic (polyphenol oxidase) browning in food and
beverages (20). However, their structure-activity relationships
have been poorly understood. Therefore, our identification of
specific compounds having the 4-substituted resorcinol skele-
ton as potent inhibitors, as outlined above, and the notion
that hydrophobic and less bulky substituents were important
for controlling the tyrosinase inhibitory effect, may lead to
the design and discovery of new tyrosinase inhibitors (Fig. 4).
The natural products and synthesized chemicals having 4-
substituted resorcinol skeletons should be reinvestigated with
regard to their roles as tyrosinase inhibitors. Furthermore,
from the chemotaxonomic point of view, specific extracts of
plants known as having flavonoids, stilbenes or other types
with 4-substituted resorcinol skeleton, for example Moraceae
(2) or Leguminosae (16), (17), are candidates for tyrosinase in-
hibitory materials. Finally it should be noted that these com-
pounds not only inhibit the tyrosinase but also have other
properties, such as resveratrol derivatives which act as an an-
tioxidant, an antimutagen and a cancer chemopreventive
agent (21).
Table 3 The tyrosinase inhibitory effects of representative 4-substi-
tuted resorcinols tested in reaction using DL-DOPA as a substrate.
Compound
IC50 (mM)
Ki (mM)
Type of
inhibition
9
90.4
20.8
17.6
19.2
6.35
3.80
17.2
47.8
9.24
8.70
13.4
8.49
5.39
11.8
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Mix
22
23
24
25
40
Kojic acid
Fig.3 Lineweaver-Burk
plots of mushroom tyro-
sinase and DL-DOPA in
the absence or presence
of 4-prenyloxyresveratrol;
&
^
Control,
4.8 mM 4-
*
prenyloxyresveratrol,
16 mM
veratrol.
4-prenyloxyres-
Fig.4 Summarized structure ± activity relationships of compounds
having 4-substituted resorcinol skeleton.

Structure-Activity Investigations
Planta Med. 66 (2000) 15
Dr. Ryuichiro Kondo
Acknowledgements
Department of Forest Products
Faculty of Agriculture
Kyushu University
Fukuoka, 812-8581
Japan
We thank Mr. T. Takeshima (Lab. of Polymer Science, Dep. of
Forest Products, Fac. of Agriculture, Kyushu University) for
providing bis(2,4-dihydroxyphenyl)methane (34). This work
was supported in part by a Grant-in-Aid for Scientific Re-
search from the Japanese Ministry of Education (Research Fel-
lowships of the Japan Society for the Promotion of Science for
Young Scientist) and the Cosmetology Research Foundation
(Tokyo, Japan).
E-mail: kondo@agr.kyushu-u.ac.jp
Fax: +81-92-642-2989
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