Natural and Synthetic Tyrosinase Inhibitors as

Antibrowning Agents: An Update

M. R. Loizzo, R. Tundis, and F. Menichini

Abstract: Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, can cause enzymatic browning in raw fruits, veg-
etables, and beverages. Browning is an undesirable reaction that is responsible for less attractive appearance and loss in
nutritional quality. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in the
food industry. This article reviews recent studies on tyrosinase inhibitors of natural and synthetic origins. The information
offered here should help food industry in developing and using potential tyrosinase inhibitors desirable efficacy and safety,
and for improving food quality.

Introduction so on (Zheng and others 2008a). Enzymatic browning represents

Appearance is one of the attributes that are considered by
consumers when they choose a food product. Among them color
is a critical determinant for the appearance of fruits, vegetables,
and crustaceans. Browning usually impairs the color attribute
together with sensory properties such as flavor and texture
(softening). However, this process is sometimes desirable, as it
can improve the sensory properties of some products such as dark
raisins and fermented tea leaves (Martinez and Whitaker 1995).
Browning occurs by 2 components: enzymatic and nonenzymatic
oxidation. Specifically, reactions of amines amino acids, peptides,
and proteins with reducing sugars and vitamin C (nonenzymatic
browning, often called Maillard reaction browning), and quinones
(enzymatic browning) cause deterioration of food during storage
and commercial or domestic processing. The loss of nutritional
quality is attributed to the destruction of essential amino acids
and a decrease in digestibility and inhibition of proteolytic and
glycolytic enzymes. The production of antinutritional and toxic
compounds may further reduce the nutritional value and possibly
the safety of foods (Mauron 1990).
Enzymatic browning results from the action of a group of
enzymes namely tyrosinase. This enzyme is widely distributed in
nature, including bacteria, fungi, higher plants (with particularly
high amounts in mushroom, banana, apple, pear, potato, avocado,
and peach), and animals (Mayer 2006). Enzymatic browning of
fruits, vegetables, and beverages takes place in the presence of
oxygen when tyrosinase and its polyphenolic substrates are mixed
after brushing, peeling, and crushing operations, which lead to
the rupture of cell structure (Hurrel and Finot 1984). The rate of
enzymatic browning depends on the concentration of tyrosinase
and phenolic substrates, oxygen availability, pH, temperature, and
MS 20111484 Submitted 12/12/2011, Accepted 3/5/2012. Authors are with the
Dept. of Pharmaceutical Sciences, Faculty of Pharmacy, Nutrition and Health Sciences,
Univ. of Calabria, 87036-I Rende (CS), Italy. Direct inquiries to author Loizzo
(E-mail: mr.loizzo@unical.it).
one of the food industry’s major problems, especially for fruits,
vegetables, and seafood products. In order to prevent browning,
use of food additives has been recognized including reducing
agents and enzyme inhibitors.
Actually, only very few enzyme inhibitors are used in the
industry due to off-flavors, food safety, and economic feasibility.
The food industry frequently uses ascorbic acid and various
forms of sulfite-containing compounds as antibrowning agents.
However, sulfite-containing compounds, in addition to causing
off-flavors, can cause allergies and, consequently, their application
on fresh-cut food products has been banned by the U.S. Food
and Drug Administration (US FDA 1986). For this reason the
food industry has introduced as antibrowning agents formulations
of ascorbic and citric acids that are, however, less effective than
sulfiting agents, since ascorbic acid is quickly consumed in the
process of reducing quinones formed by tyrosinase (Hsu and oth-
ers 1988; Santerre and others 1988). Recently, 4-hexylresorcinol
was introduced for the prevention of shrimp melanosis and for
browning control in fresh and dried fruit slices (McEvily and
Iyneger 1992). The importance of safety in the food industry
directs researchers in a constant quest for better inhibitors from
natural sources as they are largely free of any harmful side effects.
The use of alternative methods to avoid browning includes
autoclaving and blanching, whereby the food products are
immersed in a liquid at 80 to 90 ◦ C for 10 to 12 min or passed
through a forced-steam flow. These conventional processes are
inherently linked to important weight and nutritional quality
losses in the treated product (Konanayakam and Sastry 1988). One
of the alternatives that have been proposed are high hydrostatic
pressure, irradiation, pulsed electric fields, and microwave energy
(Queiroz and others 2008).
Some tyrosinase inhibitors have been discovered and com-
mented on. Kim and Uyama (2005) reviewed tyrosinase inhibitors
from both synthetic and natural sources for their industrial
importance. Likhitwitayawuid (2008) reviewed naturally occur-
ring stilbenes used as tyrosinase inhibitors not only for food


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Tyrosinase inhibitors: an update. . .
applications but also as skin whitening agents. That review article
also suggests the use of tyrosinase inhibitors as alternative insect
control agents, considering that in insects, this enzyme is essential
for the sclerotization of the exoskeleton, wound healing, and par-
asite encapsulation. Recently, Chang (2009) reviewed an update
of tyrosinase inhibitors and their inhibitory mechanisms of action.
The present review surveys and summarizes tyrosinase in-
hibitors newly discovered from natural and synthetic sources
published from 2009 until today. The mechanisms of action and
the structure-activity relationships are also discussed, wherever
possible. The knowledge offered in this review should help to
provide leads to the ultimate goal of developing new tyrosinase
inhibitors of adequate efficacy and safety for the prevention of
browning in plant-derived foods and seafood.
Enzymes
Several research papers and reviews have already been published
on the structural and kinetic aspects of the enzyme tyrosinase
(Sánchez-Ferrer and others 1995; Seo and others 2003; Garcı́a-
Molina and others 2007; Matoba and others 2006). Therefore, in
this section, we will merely introduce the biochemical characteris-
tics of the enzyme and its mechanism of action. Enzymatic brown-
ing results from the action of a group of enzymes called tyrosinase
(EC 1.14.18.1), catechol oxidase, catacholase, diphenol oxidase,
o-diphenolase, phenolase, or polyphenol oxidase. In plants both
soluble and membrane-bound tyrosinase have been described. Its
gene is encoded in the nucleus and translated in the cytoplasm; the
pro-enzyme formed is then transported to the chloroplast where
it is cleaved by a protease, producing the active form, while its
substrates are contained in the vacuole (Mayer 2006).
The term tyrosinase refers to its typical substrate, tyrosine. The
enzyme has a higher affinity for the l-isomers of the substrates and
its activities appear to have broad substrate-specificities (Rescigno
and others 2002). Almost all studies on tyrosinase inhibition
conducted so far have used mushroom tyrosinase because it
is commercially available. The best-characterized tyrosinases
are derived from Streptomyces glausescens, Neurospora crassa, and
Agaricus bisporus (Solomon and others 1996). Tyrosinase is a
multifunctional copper-containing enzyme, in which copper
is bound by 6 or 7 histidine residues and a single cysteine
residue. This enzyme possesses both monophenolase activity and
diphenolase activity. It is involved in the biosynthesis of melanin
and catalyzes the ortho-hydroxylation of tyrosine (monophenol)
to 3,4-dihydroxyphenylalanine or DOPA (o-diphenol), and the
oxidation of DOPA to dopaquinone (o-quinone) (Cooksey and
others 1997). During the browning process this o-quinone can
then be converted into brown melanin pigments through a
series of enzymatic and nonenzymatic reactions. The obtained
o-quinones are powerful electrophiles, which can be nucleophili-
cally attacked by water, other polyphenols, amino acids, peptides,
and proteins, leading to Michael-type additions. This enzymatic
browning can be prevented by trapping the o-dopaquinone
intermediate (Martinez and Whitaker 1995).
Plant Extracts and Isolated Natural Compounds
The screening for tyrosinase inhibition of the methanol extracts
prepared from the aerial parts of 33 Turkish Scutellaria species
was done by Senol and others (2010). Some Scutellaria species are
used in nutrition, in particular the young leaves of S. indica and S.
baicalensis which are cooked as a vegetable in some Asian countries,
whereas the whole plant of S. baicalensis is used as a tea substitute.
The methanol extract of S. brevibracteata subsp. subvelutina, S.

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brevibracteata subsp. brevibracteata, S. orientalis subsp. pichleri, S.
orientalis subsp. pectinata, S. orientalis subsp. carica, S. brevibracteata
subsp. pannosulo, S. albida subsp. Colchica, and S. hastifolia displayed
moderate inhibition on tyrosinase with a percentage ranging from
39.57% to 51.58% at 1000 μg/mL for S. albida subsp. colchica and
S. brevibracteata subsp. subvelutina, respectively.
The stem bark powder of Hesperethusa crenulata syn. Naringi
crenulata and Limonia acidissima, common tropical plant species in
the Indian subcontinent and Southeast Asia, has been used tradi-
tionally as a skin whitening treatment. Dichloromethane extract
exhibited the highest IC50 value of 0.546 mg/mL. This IC50 value
was approximately 60 times higher than that for kojic acid (IC50
value of 0.0009 mg/mL), a standard tyrosinase inhibitor commonly
used in cosmetic and food formulations (Wangthong and others
2010). Sapindus mukorossi, a shrub commonly known to produce
soap nuts, generally grows in tropical and sub tropical regions of
Asia. Methanol and ethyl acetate extract of these seeds have been
evaluated for their mushroom tyrosinase inhibitory activity, which
exhibited weak bioactivity with IC50 values of 17.8% and 12.3% at
10 μg/mL (Chen and others 2010). Methanolic extracts of Magno-
lia denudata and M. denudata var. purpurascens flowers were evaluated
for their tyrosinase inhibitory activities, finding IC50 values of 3.34
and 10.55 mg/mL for M. denudata and M. denudata var. purpuras-
cens, respectively, which were about 14- to 46-fold higher than that
of ascorbic acid (IC50 value of 0.23 mg/mL) (Jo and others 2011).
Polygonatum odoratum, Ampelopsis japonica, and Lindera aggregate,
used in traditional Chinese medicine, showed IC50 values of 98.4,
152.1, and 276.3 μg/mL, respectively. As compared to vitamin
C, which is widely used as an effective tyrosinase inhibitor,
all extracts and Qian-wanghong-bai-san, Qiong-yu-gao, and
San-bai-tang formulas showed smaller IC50 values against tyrosi-
nase TOGLIEREI: inhibitory activity. Moreover, as compared
with arbutin (IC50 value of 131.4 μg/mL), another known
potent tyrosinase inhibitor, extracts from San-bai-tang formula,
L. aggregate, and A. japonica had almost similar activity with
IC50 values of 80.3, 115.1, and 117.3 μg/mL, respectively (Ye
and others 2010). Methanol extract of Citrus grandis fruit tissue
inhibited tyrosinase by up to 90.8%, similar to the reference
compound kojic acid (95%), at 10 mg/mL (Wu and others 2010).
Sixteen tropical vegetables extracted with different solvents
were screened for their tyrosinase inhibitory activity. Among them
Raphanus sativus (50% propylene glycol) exhibited a percentage
of inhibition of 88.50% against tyrosinase, 78.98% was found for
Momordica charantia (methanol), 68.73% for Raphanus sativus (ethyl
acetate), and 46.57% Cymbopogon citrates (n-hexane) (Kamkaen
and others 2007).
The effects of pineapple juice (PJ), pineapple shell extract (PSE),
and rice bran extract (RBE) on the browning process that occurs
in banana slices and puree, compared with citric acid solution at
pH 3.8 (pH) and distilled water (DW), were investigated by mea-
suring the color changes in these 2 systems. RBE-treated banana
slices had lower browning value than those treated with PJ, PSE,
pH, and DW after 3 and 12 h. In fact its browning value after 12 h
was 12.05. The luminosity, expressed as L∗ values, of banana slices
treated with RBE was higher while a∗ values, which indicate the
position on the green (−) to red (+) axis, were lower than those
treated with PJ, PSE, pH, and DW after storage 12 h. RBE had
an interesting effect also in that it retarded the browning process
in banana pure where browning was lower (22.63) than when
treated with other extracts after storage for 5 h. The L∗ values of
banana puree treated with RBE were higher, whereas its a∗ values
were lower than those treated with PJ, PSE, pH, and DW after
Tyrosinase inhibitors: an update. . .
6 h. In conclusion, RBE was effectively reducing the browning
process in both systems (Theerakulkait and Sukhonthara 2008).
The extracts obtained from leaves and stems of Podocarpus elon-
gatus, P. falcatus, P. henkelii, and P. latifolius, used in traditional
medicine in Southern Africa, were investigated for their tyrosinase
inhibitory activity. The most active extract was obtained from P.
elongatus stem (EC50 value of 0.14 mg/mL), followed by P. fal-
catus leaves (EC50 value of 0.29 mg/mL) (Abdillahi and others
2011). In Podocarpus genus, amentoflavone (1) and nor- and bis-
norditerpenes have been identified. These compounds are able
to inhibit tyrosinase (Roy and others 1987; Cheng and others
2007). Moreover, in the Podocarpaceae family an effective in-
hibitor of tyrosinase, epicatechin, was identified (Kubo and others
2003). Amentoflavone (1) was identified together with naringenin
as main constituent of Inulae flos hot water extract. This extract
tested against mushroom tyrosinase using dl-DOPA as substrate
inhibited the enzyme in a dose-dependent manner with an IC50
value of 4.35 mg/mL. Kinetics analysis by the Lineweaver-Burk
plot revealed that I. flos extract act as noncompetitive inhibitor with
Ki of 1.48 mM (Wu and others 2010). The tyrosinase inhibitory
activity of I. flos could be related to the identified flavonoids since
both isolated compounds contain a resorcinol and it is well known
that this moiety is crucial for enzyme inhibition. Si and oth-
ers (2012) studied the inhibitory effects of hesperetin on mush-
room tyrosinase using inhibition kinetics and computational sim-
ulation. Hesperetin inhibited the enzyme in a dose-dependent
manner with an IC50 value of 11.25 mM. The kinetics analysis
by the double-reciprocal Lineweaver-Burk plot revealed that this
flavanone reversibly inhibited tyrosinase in a competitive-manner
with a Ki value of 4.03 mM. Hesperetin probably interacts with
the enzyme in the same way as other flavonoids through a copper
chelator activity. Through computational docking simulation stud-
ies authors hypothesized that hesperetin could bind the enzyme
through residues localized in the active site (Met280, His61, His85,
and His259). The flavonoid chrysontemin, isolated from the leaves
of Diospyros kaki, showed a weak inhibitory activity with an IC50 of
211 μM. For the same plant hyperoside, isoquercitrin, quercetin-
3-O-(2 -O-galloyl-β-d-glucopyranoside), trifolin, astragalin, and
kaempferol-3-O-(2 -O-galloyl-β-d-glucopyranoside) were iso-
lated but did not exhibit any inhibitory activity against the en-
zyme. Structure Activity Relationship (SAR) analysis evidenced
that chrysontemin, isoquercitrin, and astragalin present a glucopy-
ranoside moiety at the C-3 position of the flavonoid moiety but
this function is not indispensable for the bioactivity. On the con-
trary, the structure of isoquercitrin is different from chrysontemin
for the 4-keto group of the C ring, which seems to be responsible
for the antityrosinase activity. Moreover, it seems that the 3 ,4 -
dihydroxy groups of isoquercitrin cannot be inserted into the ac-
tive site of the enzyme. Similarly in hyperoside and quercetin-3-O-
(2 -O-galloyl-β-d-glucopyranoside) the steric hindrance effects
could hinder the entry into the active pocket. Lineweaver-Burk
plot revealed that chrysontemin acted as competitive inhibitor,
acting probably through chelation of binuclear copper localized in
the catalytic center of the enzyme (Xue and others 2011).
Salvia is a large genus distributed throughout the world.
S. cryptantha and S. cyanescens were tested for their tyrosinase
inhibitory activity, finding that it had low inhibitory activity
as compared to the reference kojic acid (Süntar and others
2011). This might presumably be related to the phytochemical
content, although these species are known to contain phenolics in
remarkable amounts. Nerya and others (2004) stated that the most
important factor in efficacy of the chalcones against tyrosinase is
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the location of the hydroxyl groups on aromatic rings, while Lee
and others (2004) reported that prenylation with isoprenyl group
or vinylation of the flavonoid molecules did not enhance tyrosinase
inhibitory activity. In fact, among prenylated compounds tested
kuwanon C (2) and sanggenon D (3) were found to possess con-
siderable inhibitory activity with IC50 values of 49.2 and 7.3 μM,
respectively. Conversely, the addition of a vinyl moiety did not
result in obtain bioactivity. Previously, Kang and others (2004)
reported the ability of rosmarinic acid and its methyl ester, isolated
from S. miltiorrhiza to inhibit mushroom tyrosinase with IC50
values of 16.8 and 21.5 μM, respectively. They found bioactivity
to be comparable to kojic acid (IC50 value of 22.4 μM). Both
compounds acted as competitive inhibitors with Ki values of 2.4 ×
10−5 and 1.5 × 10−5 M for rosmarinic acid and its methyl ester,
respectively. Successively, Lin and others (2011) reported com-
parative inhibitory activity against tyrosinase of rosmarinic acid,
methyl rosmarinate, and pedalitin (4) isolated from Rabdosia serra.
Compound 4 exhibited the most promising activity with an IC50
value of 0.28 mM followed by methyl rosmarinate. The inhibitory
effect of methyl rosmarinate was higher to that of rosmarinic acid.
SAR analysis revealed that methoxy substitution could increase
the bioactivity. Both rosmarinic acid and methyl rosmarinate were
considered as competitive inhibitors of tyrosinase, while pedalitin
was suggested to be a mixed-type inhibitor of tyrosinase.
Biofractionation of Anastatica hierochuntica, an Egyptian herbal
medicine led to the isolation of silybin A (5) and B (6), isosilybin A
(7) and B (8), kaempferol, and quercetin, which inhibited mush-
room tyrosinase with percentages of inhibition of 24.2%, <20%,
29.4%, 30.8%, 24.2%, and 45.8%, respectively, at a concentration
of 30 μM (Figure 1). (Nakashima and others 2010). Fermented
sorghum is used to prepare a popular distilled liquor called Kaoliang.
Over 200 tons of distillery residues are produced every day from
this process. The inhibitory effect of water (SWE), 70% aqueous
acetone (SAE), and methanol (SME) extracts of sorghum distillery
residue (SDR) on mushroom tyrosinase activity was investigated.
The results clearly showed higher mushroom tyrosinase inhibitory
activity of SME (IC50 value 0.56 mg/mL) followed by SWE
(IC50 value 0.76 mg/mL) and SAE (IC50 value 0.82 mg/mL).
The SME extract also had the highest total contents of phenolics,
flavonoids, anthocyanins, and tannins, and for this reason it was
the most active (Wang and others 2011a). The same research
group analyzed the effect of aqueous extract of green asparagus
(AGA) against mushroom tyrosinase. The results showed that
AGA inhibited tyrosinase with an IC50 value of 1.21 mg/mL in
a mixed-type inhibition. The High-Pressure Liquid Chromatog-
raphy (HPLC) analysis revealed the presence of rutin, quercetin,
kaempferol, and isorhamnetin, known as tyrosinase inhibitors
(Wang and others 2011b). Previously, Jeong and Shim (2004)
demonstrated that quercetin, isolated from Zanthoxylum piperitum,
showed significant mushroom tyrosinase inhibitory activity with
an IC50 value of 3.8 μg/mL and acting as a competitive inhibitor.
Broussonetia papyrifera, commonly known as paper mulberry,
is a deciduous tree or shrub, which grows naturally in Asia and
Pacific areas. From the chloroform-soluble extract of B. papyrifera
twigs 3,5,7,4 -tetrahydroxy-30-(2-hydroxy-3-methylbut-3-enyl)
flavone (9), uralenol (10), quercetin, isolicoflavonol (11),
papyriflavonol A (12), broussoflavonol F (13), 5,7,30,50-
tetrahydroxyflavanone (14), luteolin, isoliquiritigenin, brous-
sochalcone A (15), and 5,7,30,40-tetrahydroxy-3-methoxyflavone
(16) were isolated. An evaluation of tyrosinase inhibitory activity
showed IC50 values of 96.6, 49.5, 57.8, and 82.3 μM for 9, 10,
and 13, respectively (Zheng and others 2008a).

c 2012 Institute of Food Technologists®
Tyrosinase inhibitors: an update. . .
OH
HO
HO O HO
O
OH
OH
OH
HO O
1
OH O MeO
HO
OH
O CH2OH
HO O OMe
O 6
OH OH
OH O 5
OH
O HO
OMe
HO O
O CH2OH
OH 7
OH O
Figure 1–Structures of compounds 1–8.
Jackfruit (Artocarpus heterophyllus) is an exotic fruit grown in
tropical climates. The various parts of the jackfruit tree have also
been reported as valuable ingredients in the preparation of dif-
ferent Ayurvedic and Yunani medicines. Recently, some of the
prenylated flavonoids were isolated from the A. heterophyllus and
A. incisus wood as antibrowning agents (Zheng and others 2008b,
2009). Among them norartocarpetin (17), artocarpesin (18), step-
pogenin (19), isoartocarpesin (20), and artocarpanone (21) showed
promising IC50 values of 0.46, 0.52, 0.57, 0.66, and 1.54 μM,
respectively, similar to glabridin (IC50 value of 0.57 μM), a well-
known tyrosinase inhibitor isolated from licorice roots that has
been extensively used in expensive cosmetic products (Figure 2).
Bioactive guided fractionation of A. heterophyllus wood extract
led to the isolation 3-prenyl luteolin which showed tyrosinase in-
hibitory activity with IC50 of 76.3 μM (Arung and others 2010).
SAR analysis revealed that luteolin act as a substrate of tyrosinase
since it was easily oxidized by the enzyme due to the presence of a
catechol moiety at the B ring. Probably the steric hindrance of the
prenyl substituent at the C-3 position in 3-prenyl luteolin prevents
the binding of the catechol moiety at the B ring to the active site.
This behavior is similar also for quercetin (Kubo and others 2004).
Ninety-one central Argentina native plant extracts were
tested for their tyrosinase inhibitory activity (Chiari and others
2010). Among them Achyrocline satureioides, Artemisia verlotiorum,
Cotoneaster glaucophylla, Dalea elegans, Flourensia campestris, Jodina
rhombifolia, Kageneckia lanceolata, Lepechinia floribunda, Lepe-
chinia meyenii, Lithrea molleoides, Porlieria microphylla, Pterocaulon
alopecuroides, Ruprechtia apetala, Senna aphylla, Sida rhombifolia,

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HO O OH
HO OH
O
O OH O
O OH
HO
2 3
OH OH
O
OH
O CH2OH
O 4
HO O OMe
O
OH OH
OH O
OH
O
OMe
O
O CH2OH
OH
8
OH O
Solanum argentinum, Tagetes minuta, and Thalictrum decipiens
exhibited more than 90% inhibition of tyrosinase monophenolase
activity at 1000 μg/mL. In particular, D. elegans and L. molleoides
exhibited the most interesting activity with IC50 values of 0.48
and 3.77 μg/mL, respectively. Both extracts were also able to
inhibit diphenolase activity but with higher IC50 values. L.
molleoides ethanolic extract biofractionation led to the isolation of
a resorcinol analog, (Z,Z)-5-(trideca-4,7-dienyl)-resorcinol, that
showed tyrosinase inhibitory activity (IC50 values 0.49 μg/mL).
(Z,Z)-5-(trideca-4,7-dienyl)-resorcinol was 37 times more active
in monophenolase inhibitory activity than kojic acid used as a
reference. Following their previous result on D. elegans, Chiari
and others (2011) reported the isolation of dalenin (22) as the
active compound with an IC50 value of 0.26 μM using l-tyrosine
as substrate. Dalenin (22) acted as reversible inhibitor of the
enzyme and that it was a mixed-I-type inhibitor with l-tyrosine
as substrate. Dalenin action could be ascribed not only to its
substituents but also to its ability in chelating the enzyme. SAR
analysis revealed that the free hydroxyl functions in ring B are not
the only substitution group responsible for the remarkable activity
of compound 22. The presence of the 2,2-dimethylchromene ring
system substituted at the 6 to 7-position may also be important for
its inhibitory activity, since the flavanone steppogenin (19), which
has a similar structure but lacks the pyrane ring, exhibited lower
bioactivity. Moreover, the chromene ring not only influences the
potency of inhibitions but also the method, since steppogenin
(19) acts as a competitive inhibitor with both oxidative reactions
catalyzed by tyrosinase (Jeong and others 2009). The lack of
Vol. 11, 2012 r Comprehensive Reviews in Food Science and Food Safety 381
Tyrosinase inhibitors: an update. . .

R6
OH
R5
R2
OH HO O
R4 HO O
OH OH
R1 R3
HO O
OH O
OH O
OH 10 R1= R2= H, R3= R5= R6= OH, R4= prenyl
11 R1= R2= R6= H, R3= R5= OH, R4= prenyl 14
OH O
12 R1= R4= prenyl, R2= H, R3= R5= R6= OH
9 13 R2= R4= prenyl, R1= R6= H, R3= R5= OH
OH 16 R1= R2= R6= H, R4= R5= OH, R3= OMe
OH
R HO OH
HO HO OH
HO O
RO O

OH O R
OH O
15 R= prenyl OH O
OH 17 R= H 19 R= H
21 R=Me
O O 18 R=
OH
20 R=
OH O
22

Figure 2–Structures of compounds 9–22.

the 2,3-unsaturation pattern seems to make compound 22 more
active, since cycloartocarpesin (23) that was characterized by the
same structure as dalenin (22) but with the above-mentioned
chemical feature, did not show any inhibitory activity.
Recently, Zheng and others (2011) analyzed the Cudrania
cochinchinensis stem and root extract finding IC50 values of 36.3
and 56.2 μg/mL, for stem and root, respectively. (±) 2,3-cis-
Dihydromorin, 2,3-trans-dihydromorin, and oxyresveratrol were
isolated and tested finding strong IC50 values of 31.1, 21.1, and
2.33 μM. Other identified constituents such as quercetin-7-O-β-
d-glucoside, kaempferol-7-O-β glucopyranoside, and morin-7-
O-β-d-glucoside showed moderate inhibitory activity. The num-
ber and position of hydroxyl groups play a crucial role in the in-
hibitory activity especially if they are at 2 -position and 4 -position
of the B ring. Moreover, trans-configuration exhibited slightly
stronger tyrosinase inhibitory activity than that of cis-configuration
as observed for 2,3-trans-dihydromorin and 2,3-cis-dihydromorin.
The ethyl acetate extract of the soygerm koji fermented
with Aspergillus oryzae (BCRC 32288) showed an IC50 value of
0.19 mg/mL against tyrosinase. Bioctivity-guided partionation
led to the isolation of 6,7,4 -trihydroxyisoflavone, 7,8,4 -
trihydroxyisoflavone, 5,7,8,4 -tetrahydroxyisoflavone, daidzein,
glycitein, daidzin, and genistin. 6,7,4 -Trihydroxyisoflavone
(24), daidzein, glycitein, daidzin, and genistin inhibited only
the monophenolase activity of mushroom tyrosinase with IC50
values of 0.009, 0.203, 0.218, 0.267, and 0.343 mM, respectively.
Kinetic study by the Lineweaver-Burk method showed that this
isoflavones acted competitively. 7,8,4 -Trihydroxyisoflavone and
5,7,8,4 -tetrahydroxyisoflavone inhibited both monophenolase
and diphenolase activities of tyrosinase and acted as irreversible
inhibitors. This study also confirmed that the number of hydroxyl
groups in the A-ring of the isoflavone structure could affect the
biaoactivity. For example, in compound 24 the presence of hy-
droxyl groups at both C6 and C7 position in the A-ring increased
both inhibitory activity and affinity for the enzyme with respect to
the isoflavones with only one hydroxyl group at the C7 position
such as daidzein and glycitein or without any hydroxyl group
such as daidzin and genistin. Moreover, the presence of hydroxyl
groups at both C7 and C8 position determined the transformation
from reversible to irreversible inhibitors. At the same time it is of
certain interest to note that the presence of a hydroxyl group at
the C7 position is not determinant for the potency (daidzein and
glycitein vs daidzin and genistin). The antibrowning activity of soy
isoflavones is of interest for food industry since these compounds
and their derivatives occur in many fermented soybean foods in
amounts of several mg/g, which is much higher than the concen-
tration category, useful to block the tyrosinase (Chang and others
2007). From the mung bean (Vigna radiatae L.) flavones vitexin
and isovitexin were isolated. These flavones exhibited a low
mushroom tyrosinase inhibitory activities, with IC50 values of 6.3
and 5.6 mg/mL, respectively (Yao and others 2011). Sophorafla-
vanone G (25), kurarinone (26), and kurarinol (27) were isolated
from Sophora and tested for their tyrosinase inhibitory activity,
finding IC50 values of 4.7, 2.2, and 0.1 μM, respectively (Ryu and

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Tyrosinase inhibitors: an update. . .
OH
HO O OH
O O
OH
OH O OH
OH O
24
23
OH
HO
HO
HO O
OH
OMe O
OMe O
26
Figure 3–Structures of compounds 23–29.
others 2008). Through SAR analysis the authors identified in the
2 ,4 -dihydroxylated B-ring system the portion of the structure
responsible of bioactivity (Khatib and others 2005). However,
compound 27, which possesses a hydroxylated lavandulyl function,
is 50-times more potent than lavandulylated flavanones 25 and 26.
Furthermore, kurarinol (27) is 200 times more potent than
norartocarpetin, a structurally related flavanoid which does not
contain the lavandulyl moiety (Kittisak and others 2000). Kinetic
analysis revealed that both sophoraflavanone G (25) and kurari-
none (26) are noncompetitive inhibitors of mushroom tyrosinase,
while kurarinol (27) was a competitive inhibitor. The roots of
Morus nigra are an ingredient in skin whitening cosmetics. Zheng
and others (2010a) analyzed several phytochemicals belonging
to the flavonoids such as stilbene glycosides; 2-arylbenzofuran
derivatives, and coumarin glycosides. Among the chalcones,
2,4,2 ,4 -tetrahydroxychalcone (28) and morachalcone A (29)
were the 2 strongest inhibitors with IC50 values of 0.062 and
0.14 μM, respectively (Figure 3). A lower bioactivity was
observed with 5 -geranyl-5,7,2 ,4 -tetrahydroxyflavone that
exhibited an IC50 of 37.09 μM, which is a lower activity than that
of norartocarpetin and artocarpesin, suggesting that the presence
of an isoprenyl/geranyl group in the B-ring of the flavonoid
skeleton significantly modifies the enzyme inhibitory activity.
The presence of the isoprenyl group on the E ring modifies the
bioactivity. In fact, the presence of 2 isoprenyl groups at the
24-position on the E ring, as in kuwanon h (IC50 of 10.34 μM),
led to much more potent activity compound to kuwanon G
(IC50 > 200 μM), which has only one isoprenyl group in the same
position. Kuwanon U, an isogeranyl flavanone, showed much
lower inhibitory activity than kuwanon E (30) with IC50 values >
200 and 77.99 μM, respectively. This observation suggested that
the substitution of a methyl group at the 4 -OH group on the
B-ring, as in kuwanon U, compromises the bioactivity. Among
the tested compounds were also 3 stylbene glycosides that inihib-
ited the enzyme: oxyresveratrol-3 -O-β-d-glucopyranoside (IC50
value of 1.64 μM), oxyresveratrol-2-O-β-d-glucopyranoside
(IC50 value of 29.75 μM), and mulberroside A (IC50 value >
200 μM). SAR analysis revealed that glycosidation at the 2 or
4-position substantially weakened activity to a much larger extent

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HO O
OH
OH O
25
HO OH
OH
HO
O
OH R
OH O
28 R= H
27 29 R= isoprenyl
than at the 3 or 5 position. Among the isoprenyl-substituted
2-arylbenzofuran derivatives, moracin N exhibited the highest
inhibitory activity with an IC50 value of 30.52 μM. In this
compound the isoprenyl group remains intact, which might
contribute to its higher tyrosinase inhibitory activity relative to
moracin O (IC50 value of 93.58 μM) in which the isoprenyl group
forms a 5-membered ring with the -OH group at the 6-position.
The ethanolic extract of Greyia flanaganii leaves showed an
interesting tyrosinase inhibitory activity (IC50 of 32.62 μg/mL).
Following this observation, bioassay-guided fractionation was
done which led to the isolation of only one bioactive constituent,
2 ,4 ,6 -trihydroxydihydrochalcone; it showed the highest activity
with an IC50 value of 69.15 μM. (Mapunya and others 2011).
From Anacardium occidentale nuts cardol triene was isolated.
This compound reveal strong irreversible competitive tyrosinase
inhibitory activity (IC50 value of 22.5 μM) (Zhuang and
others 2010). The SAR analysis revealed that the presence of a
resorcinol-moiety could drastically enhance its enzyme inhibitory
activity. The inhibitory kinetics studies showed that cardol triene
is a mixed-type inhibitor.
The tyrosinase inhibitory activity of ethanolic extract of
Hibiscus cannabinus leaf was evaluated before and after subjecting it
to far-infrared (FIR) irradiation. Several reports have shown the
potential of FIR treatments for enhancing the activity levels and
the content of functional components in food products (Lee and
others 2005, 2006; Eom and others 2009). Purification of extract
led to the isolation of kaempferitrin as the main component.
Prior to FIR irradiation, no tyrosinase inhibitory activity was
detected. On the contrary, after FIR treatement (for 1 h at 60
◦
C) significant tyrosinase inhibitory activity was observed (IC50
value of 3500 ppm). HPLC analysis of this extract identified
kaempferol, afzelin, and α-rhamnoisorobin as derhamnosylation
products. Among them kaempferol exhibited an IC50 value of
171.4 μM (Rho and others 2010a).
Mango (Mangifera indica) is the most cultivated fruit in Thailand.
Several studies have shown that mango seed kernels contain vari-
ous phenolic compounds including gallotannins, epicatechin, and
condensed tannin-related polyphenols (Arogba 2000; Puravankara
and others 2000; Abdalla and others 2007). Refluxing in acidified
Tyrosinase inhibitors: an update. . .
ethanol extract of sun-dried mango seed kernels was characterized
by the highest total phenolics content and tyrosinase inhibitory
activity with an ID50 value of 4.13 mg/mL. This extract acts as a
competitive inhibitor of the enzyme. Moreover, it was also shown
to be a good chelator of copper, which is the metal at the center
of the active site of tyrosinase (Maisuthisakul and Gordon 2009).
Recently, Ko and others (2011) investigated the potential
tyrosinase inhibitory activity of 70% ethanol extract obtained
from the branches of Distylium racemosum. EtOAc and n-butanol
fraction showed the stronger mushroom tyrosinase inhibitory
activity, using l-tyrosine as the substrate with IC50 values of 27.1
and 29.6 μg/mL. From the EtOAc fraction 20 compounds were
isolated. Among them gallocatechin showed a strong tyrosinase
inhibitory activity with an IC50 value of 4.8 μg/mL. Other
constituents such as epi-gallocatechin gallate, methyl gallate, and
quercitrin exhibited interesting IC50 values of 30.2, 40.5, and
37.3 μg/mL, respectively. The extracts of dog rose (Rosa canina)
hips and pomegranate (Punica granatum) arils were assayed for
inhibition of tyrosinase derived from mushrooms and vegetables
and polyphenol oxidase activity. In addition to the in vitro studies,
melanosis in foods such as artichokes, mushrooms, and pear
juice were evaluated. The results revealed that dog rose hip
extract was more effective than pomegranate aril extract and
this bioactivity could be related to the total phenolics content
since in the pomegranate extract it was 1.45 mg gallic acid
equivalent/mL, while in dog rose extracts it was 9.16 mg gallic
acid equivalent/mL. Dog rose extract analyzed by HPLC showed
a high content of epigallocatechin (8 mM), which has been
reported as a competitive inhibitor of tyrosinase (Zocca and
others 2011). Another flavanol that possesses similar properties
is epigallocatechin gallate found in pomegranate extract, it
contained an average of 335 mg/L (0.7 mM) of epigallocatechin
gallate, a value higher than the IC50 value (0.034 mM) reported by
Kim and Uyama (2005). The same research group reported also an
IC50 value of 0.017 mM for epicatechin gallate when inhibiting
tyrosinase; in the dog rose extract, epicatechin gallate was found to
have a concentration of 230 mg/L (0.52 mM). Another phenolic
compound found in dog rose extract that possesses antipolyphenol
oxidase/tyrosinase activity is p-hydroxybenzoic acid (485 mg/L,
3.5 mM). The hydroxybenzoic acids also include ellagic acid,
which has antityrosinase activity that was detected only in the dog
rose extract (177 mg/L) (Yoshimura and others 2005; Zocca and
others 2011). Previously, Lin and others (2010) had reported on
the inhibitory activities of p-propylbenzoic acid, p-butylbenzoic
acid, p-pentylbenzoic acid, p-hexylbenzoic acid, p-heptylbenzoic
acid, and p-octylbenzoic acid on potato polyphenol oxidase,
finding IC50 values from 0.047 to 0.213 mM for p-octylbenzoic
acid, and p-propylbenzoic acid, respectively. All benzoic acids are
reversible inhibitors, but the inhibitory types were determined
to be noncompetitive. The result of the inhibitory type lead the
authors to hypothesize that the inhibitors attach to the enzyme
at a site different from the active site and hinder the binding of
substrate to the enzyme through steric hindrance or by changing
the protein conformation. On the basis of their observations the
authors speculated that when substrate, it can bind the enzyme and
will induce a new hydrophobic pocket in the enzyme-substrate
complex, and the para-position hydrocarbon chain can then
be inserted into the pocket. Among these tested compounds,
p-octylbenzoic acid was the most potent inhibitor, suggesting that
the hydrophobic pocket accepts the 8 hydrocarbon chain well.
The results pointed out that the inhibitor could be embraced by
the enzyme hydrophobic pocket. This mechanism, in addition,
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is probably involved in the tyrosinase inhibitory activity of p-
hydroxybenzoic acid and methyl p-hydroxybenzoate isolated from
the branches of Ficus erecta var. sieboldii. Both compounds showed
promising bioactivity with IC50 values of 0.98 and 0.66 mM for
p-hydroxybenzoic acid and methyl p-hydroxybenzoate, respec-
tively. Kinetics analysis by the Lineweaver-Burk plot indicated
that both compounds were competitive inhibitors of diphenolase
of mushroom tyrosinase (Park and others 2011).
Zocca and others (2010) reported the ability of processing water
prepared by cooking Brassica oleracea leaves to inhibit tyrosinase. In
particular, for mushroom tyrosinase the IC50 value was 200 μL of
processing water. This water contained high total sulfur content
(0.218 g/L) together with phytochemicals such as glucosinolates,
phenols, antocyanins, and organic acids and is known to inhibit
the browning process Rojas-Graü and others 2008; Volden
and others 2009). Similar results were previously obtained with
another member of the Brassicaceae family, Barbarea orthocerus,
which contains the tyrosinase inhibitor barbarin showed an IC50
value of 4.2 × 10−5 M against mushroom tyrosinase (Seo and
others 1999). Processing water was also freeze-dried in order to
increase its utility and effectiveness. However, this process resulted
in a loss of bioactivity, probably due to the stresses of the process,
which can result in a degradation of biomolecules as reported by
Kamath (2006). The addition of ascorbic acid to processing water
resulted in complete inhibition of grape polyphenoloxidases, an
effect which was 34% stronger than the acid alone. One possible
mechanism for this synergistic effect is that processing water may
specifically interact with polyphenoloxidases rendering it unable
to catalyze the enzymatic reaction, while ascorbic acid reduces
the quinones generated by polyphenoloxidases.
From Ecklonia cava, a brown alga largely used as a food
ingredient, dieckol was isolated. This phlorotannin is able to
inhibit mushroom tyrosinase in a dose-dependent manner (IC50
value of 20 μM). The inhibition kinetics revealed that dieckol
behaved as a noncompetitive inhibitor. Molecular modeling
studies found that this compound could interact with residues
His208, Met215, and Gly46 in the active site of the enzyme that
were more important than those of compound arbutin (His208,
Gly216, and Asn205), and were the main contributors to the
receptor-ligand interaction (Kang and others 2012).
Crustaceans are widely consumed all over the world because of
their delicious and nutritional value. Shrimp and shrimp products
of Thailand are well known for their long-standing excellent
reputation worldwide, owing to outstanding quality, freshness,
variety, and taste (Rattanasatheirn and others 2008). Pacific white
shrimp (Litopenaeus vannamei) is an important commercial species
characterized by a very short shelf-life span, due to melanosis.
To maintain the quality and to avoid melanosis of shrimp or
other crustaceans, sulfite and 4-hexylresorcinol are widely used
(Martinez-Alverez and others 2005). Shrimp treated with 5 g/L
of green tea ethanolic extract, with prior chlorophyll removal,
brought about lower melanosis, compared with the control, and
showed similar scores to those treated with sodium metabisulfite.
The main flavonoids present in green tea include catechins (flavan-
3-ols) (Cabrera and others 2006). Catechin probably acted as a
competitive inhibitor for polyphenoloxidase because of its struc-
tural similarity with the substrate (Nirmal and Benjakul 2009).
Furthermore, the ethanolic green tea extract, with prior chloro-
phyll removal, had no adverse impact on the sensory attributes
of the treated shrimp (Nirmal and Benjakul, 2011a). On the
cephalothorax of Pacific white shrimp mimosine (β-(3-hydroxy-
4-pyridon-1-yl)-l-alanine) obtained from Leucaena leucocephala

c 2012 Institute of Food Technologists®
Tyrosinase inhibitors: an update. . .
O
OH
30
OMe
HO OH
O OH
MeO
O O
33
Figure 4–Structures of compounds 30–34.
was investigated by Nirmal and Benjakul (2011b). This nonprotein
amino acid showed a dose-dependent manner inhibitory activity
towardpolyphenoloxidase with an apparent molecular weight of
210 kDa. Kinetics analysis by Lineweaver-Burk plot indicated that
mimosine could bind both the enzyme and the enzyme-substrate
complex, but with different affinities. Moreover, it showed copper
reduction and chelating capacity in a dose-dependent manner.
Mimosine could react with the intermediate browning product,
thereby rendering lower red-brown coloring formation.
From Saccharum officinarum processing desugared sugar cane
extract (DSE) was obtained and fractionated (Chung and others
2011). Among other isolated constituents lariciresinol 4-O-β-d-
glucoside and threo-guaiacylglycerol-7-O-β-d-glucopyranoside
exhibited a promising activity with IC50 values of 42.59 and
57.72 μM. SAR analysis revealed that active compounds are char-
acterized by the presence of a free hydroxyl group at 4-position in
the aromatic ring. At the same time the presence of free carboxylic
groups that could be forming internal hydrogen binding to the
free hydroxyl groups reduced drastically the bioactivity.
A perusal analysis of literature revealed the presence of
several oxindole alkaloids isolated from Isatis costata as tyrosinase
inhibitors. Costinones A, B, isatinones A, isatinones B, indirubin,
and trisindoline exhibited a significant activity with IC50 values
ranging from 7.21 to 17.34 μM (Ahmad and others 2010).
Thymol and carvacrol are members of monoterpene phenol
class and are the main constituents of thyme oil. Both compounds
are known as antioxidants and were used as additives in protecting
food qualities. The potential use of thyme oil was analyzed using
mushroom tyrosinase and l-tyrosine as substrate. As well as the oil
thymol and carvacrol both exhibited a concentration-dependent
inhibitory activity against dopachrome formation and oxygen
consumption. SAR analysis revealed that replacement of the
hydroxyl group with the methoxy group, drastically reduced the
effect on the enzyme suggesting the crucial role of hydroxyl group
in the enzyme interaction. Interestingly, another study suggested
that thymol inhibits the redox reaction between dopaquinone and
leukodopachrome instead of enzymatic reaction. In particular, thy-
mol successfully inhibited oxidation of l-DOPA to dopaquinone,
coupled with reduction of p-benzoquinone. Hence, the suppres-

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OH
HN R
O
N
H 31 R= H
32 R= OMe
HO OH
HO O
O O
HO
O
MeO OMe 34
OH
sion of dopachrome formation by thymol is due to the inhibition
of conversion of leukodopachrome to dopachrome. On the basis
of results the authors conclude that the antibrowning effect of
thymol is due to the inhibition of the redox reaction without any
interaction with the enzyme (Satooka and Kubo 2011).
Far Eastern sea cucumber (Stichopus japonicas) extracts were
screened for their tyrosinase inhibition using different analytical
methods (Husni and others 2011). The 70% ethanolic extract
(IC50 value of 0.49 to 0.61 mg/mL) was more capable in
inhibiting tyrosinase activity than the water extract (IC50 value
1.80 to 1.99 mg/mL). Lower IC50 values were seen with
the spectrophotometric method. Kinetic analysis revealed that
extracts are reversible and mixed-type inhibitors. Through
bioactivity-guided fractionation ethyl-α-d-glucopyranoside (IC50
value of 0.19 mg/mL) and adenosine (IC50 value of 0.13 mg/mL)
were identified as bioactive constituents.
Sasa quelpaertensis dried leaves have been used to make a leaf
tea. Bioactivity-guided fractionation of S. quelpaertensis ethyl
acetate fraction led to the isolation of N-p-coumaroylserotonin
(31) and N-feruloylserotonin (32) that showed strong tyrosinase
inhibitory activity with IC50 values of 0.027 mM and 0.026 mM,
respectively. Interesting activity was also obtained with 3-O-p-
coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone
(33, IC50 value of 0.055 mM) and 3-O-p-coumaroyl-1-(4-
hydroxy-3,5-dimethoxyphenyl)-1-O-β-glucopyranosylpropanol
(34, IC50 value of 0.053 mM) considering that arbutin used as
the control showed an IC50 value of 0.048 mM (Sultana and Lee
2009) (Figure 4).
Arabinose is an aldopentose having both sugar and aldehyde
groups in its structure. This compound significantly inhibited
tyrosinase with IC50 0.1 mM, and this was accompanied by
conformational changes in enzyme structure. Arabinose induced
mixed-type inhibition with Ki value of 0.22 mM. Measurements
of intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding
fluorescence showed that arabinose induced tyrosinase to unfold
and expose inner hydrophobic regions. Computational docking
simulation analysis revealed that this inhibitor could interact with
the catalytic site through residues His61, Asn260, and Met280
(Hu and others 2012) (Table 1).
Tyrosinase inhibitors: an update. . .

Table 1–Plant extract with tyrosinase inhibitory activity.

Extracts Part of plant Extraction solvent IC50 -ID50-EC50

S. albida subsp. colchica Aerial parts MeOH 39.57% (at 1000 μg/mL)
S. brevibracteata subsp. subvelutina Aerial parts MeOH 51.58% (at 1000 μg/mL)
Hesperethusa crenulata Stem bark CH2 Cl2 0.546 mg/mL
Sapindus mukorossi Seeds MeOH 17.8% (at 10 μg/mL)
Sapindus mukorossi Seeds EtOAc 12.3% (at 10 μg/mL)
Magnolia denudate Flowers MeOH 3.34 mg/mL
Magnolia denudata var. purpurascens Flowers MeOH 10.55 mg/mL
Mangifera indica Seed kernels EtOH 4.13 mg/mL
Podocarpus elongatus Stem MeOH 0.14 mg/mL
Podocarpus falcatus Leaves MeOH 0.29 mg/mL
Polygonatum odoratum Rhizomes 30% EtOH 98.4 μg/mL
Ampelopsis japonica Roots 30% EtOH 152.1 μg/mL
Lindera aggregata Leaves 30% EtOH 276.3 μg/mL
Citrus grandis Fruit tissue MeOH 90.8 (at 10 mg/mL)
Stichopus japonicas Body without visceral organ and fluids 70% EtOH 0.49–0.61 mg/mL
Stichopus japonicas Body without visceral organ and fluids H2 O 1.80–1.99 mg/mL
Raphanus sativus Whole plant 50% Propylene glycol 88.50% (at 5 mg/well)
Raphanus sativus Whole plant EtOAc 68.73% (at 5 mg/well)
Momordica charantia Whole plant MeOH 78.98% (at 5 mg/well)
Cymbopogon citrates Whole plant n-Hexane 46.57% (at 5 mg/well)
Brassica oleracea Leaves Processing H2 O 200 μL
Dalea elegans Aerial parts EtOH 0.48 μg/mL
Lithrea molleoides Aerial parts EtOH 3.77 μg/mL
Greyia flanaganii Leaves EtOH 32.62 μg/mL
Cudrania cochinchinensis Stem MeOH 36.3 μg/mL
Cudrania cochinchinensis Roots MeOH 56.2 μg/mL
Soygerm koji fermented with Aspergillus oryzae BCRC 32288 Seeds EtOAc 0.19 mg/mL
Distylium racemosum Branches EtOAc 27.1 μg/mL
Distylium racemosum Branches n-BuOH 29.6 μg/mL
Inulae Flos Aerial parts (Tea) Hot H2 O 4.35 mg/mL

Synthetic Compounds hydroxycinnamic acid and α-cyano-4-hydroxycinnamic acid acted

It is well known that tyrosinase can be inhibited by aromatic
acids (Robit and others 1997). The effects of cinnamic acid and
its derivatives on the activity of mushroom tyrosinase have been
studied by Shi and others (2005). All these compounds strongly
inhibited the diphenolase activity of mushroom tyrosinase with
IC50 values of 2.10, 0.50, and 0.42 mM, for cinnamic acid, 4-
hydroxycinnamic acid, and 4-methoxycinnamic acid, respectively.
In particular, cinnamic acid and 4-methoxycinnamic acid were
noncompetitive inhibitors (Ki 1.994 and 0.458 mM, respec-
tively), while the 4-hydroxycinnamic acid acted as competitive
inhibitor with Ki 0.244 mM. Qiu and others (2009) analyzed the
effects of the cinnamic acid analog α-cyano-4-hydroxycinnamic
acid on both the monophenolase and diphenolase activity of
mushroom tyrosinase. For the monophenolase activity, when the
concentration of this analog reached 80 μM, the lag time was
lengthened from 20 to 150 s and the steady-state activity was
lost by about 75%. The IC50 value was estimated to be 48 μM.
For the diphenolase activity, the inhibitory effect of this analog
was lower (IC50 value of 2.17 mM). The kinetic analysis revealed
that this analog acted on the diphenolase activity as reversible and
competitive inhibitors. Because the π-conjugated framework of
hydroxycinnamic acid is much smaller than that of this analog, this
compound could easily bind to the enzyme active site. Moreover,
the presence of the cyano-group on this analog makes the binding
with the active site of tyrosinase even more difficult. The IC50 val-
ues of 4-hydroxycinnamic acid and α-cyano-4-hydroxycinnamic
acid for diphenolase activity were 0.50 and 2.17 mM, respectively.
The binding affinity of α-cyano-4-hydroxycinnamic acid to
the diphenolase was not as tight as that of 4-hydroxycinnamic
acid probably due to the presence of the cyano-group and the
ethylenic linkage in the α-cyano-4-hydroxycinnamic acid struc-
ture. So the inhibition of α-cyano-4-hydroxycinnamic acid on
the diphenolase is weaker than 4-hydroxycinnamic acid. Both 4-
on the diphenolase activity of tyrosinase as competitive inhibitors.
Kojic acid is largely used as a food additive to prevent
enzymatic browning. On the basis of this evidence a series
of kojic acid derivatives was synthetized. Among them, 2-
(cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one (35) showed
the most potent activity (IC50 value of 0.087 μM) followed by
2-(pentylthiomethyl)-5-hydroxy-4H-pyran-4-one with an IC50
value of 0.097 μM (Rho and others 2010b).
A series of oxadiazole and triazolothiadiazole derivatives were
synthesized and tested as mushroom tyrosinase inhibitors (Lam
and others 2010). Among them, 5 derivatives showed high tyrosi-
nase inhibition with IC50 values ranging from 0.87 to 1.49 μM.
SAR analysis attributed an important role to the cycloamine moi-
ety attached at N-3 of the oxadiazole ring (piperazine > piperi-
dine > pyrrolidine > morpholine > methylpiperazines). Ghani
and Ullah (2010) projected a series of 1,3,4-thiadiazole-2(3H)-
thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-
5(4H)-thiones, and substituted hydrazides. The Ki values of thia-
diazoles, triazoles, oxadiazoles, and substituted hydrazides were in
the range of 0.19 to 5.2 μM, 1.01 to2.4 μM, 1.35 to 69.4 μM, and
49 to 177.2 μM, respectively. 1,3,4-Thiadiazole-2(3H)-thiones
were found to be the most potent inhibitors. Among them the
most potent was the compound containing a 5-(4-hydroxyphenyl)
substitution. The tyrosinase inhibitory activity was drastically re-
duced when the hydroxyl group on the 4-position of the phenyl
ring was removed. Two more substitutions at the same position of
the phenyl ring with hydrophobic groups yielded better inhibitors.
The inhibitory activities of triazoles were comparable with those
of some inhibitors of the thiadiazole and oxadiazole series. In the
1,3,4-oxadiazole-2(3H)-thiones series, the compound character-
ized by a 4-benzyloxyphenyl group on the oxadiazole ring was
found to be the most potent inhibitor (K i value of 1.35 μM).
Comparison of this compound with its positional isomer showed

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Tyrosinase inhibitors: an update. . .
a 3-fold lower activity. Substitution of a 4-hydroxyphenyl group at
5-position of the oxadiazole ring was the second most potent in-
hibitor in this series. SAR analysis revealed that both the absence of
a hydroxyl group from the phenyl ring and the presence of a cyclo-
hexyl ring as a substituent at the 5-position drastically reduced the
enzyme inhibitory activity. This behavior was similar to what was
observed earlier in the thiadiazole and triazole series in which the
presence of a 5-(4-hydroxy) phenyl group increased the tyrosinase
inhibitory activity. However, a comparison of structures indicated
that the potency varied depending on the class of compounds
and the type of substitutions. Hydroxy- and/or alkoxy-substituted
phenyl-benzo[d]thiazole derivatives were recently synthesized and
evaluated for their ability to inhibit mushroom tyrosinase by Ha
and others (2011). Among them, compounds 36 and 37 exhibited
much higher tyrosinase inhibition activity (45.36% to 73.07% and
49.94% to 94.17% tested at 0.01 to 20 μM, respectively) than the
reference compound kojic acid (9.29% to 50.80% tested at 1.25 to
20 μM). The kinetic behaviors of the enzyme in the presence of
both compounds were investigated. Using compound 36 at a con-
centration of 1.25 and 20 μM the enzyme showed Km values of
1.02 and 5.65 mM, respectively, and the same Vmax value (1.54 ×
10−2 ). While in the presence of compound 37, at the same con-
centration the enzyme exhibited Km values of 1.16 and 1.46 mM,
respectively, and the same Vmax value (1.37 × 10−2 ). This en-
zyme behavior suggested that both compounds are competitive
inhibitors of mushroom tyrosinase. Recently, Ha and others (2012)
reported the synthesis and the biological evaluation of a series of
2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives.
Compounds 38 and 39 exhibited around 40% of inhibition at
20 mM similar to that of kojic acid. SAR analysis revealed that
compound 38 (an hydroxyl group at 3-position and a methoxyl
group at 4-position on the phenyl ring) has a similar structure
to DOPA while compound 39 (an hydroxyl group at 4-position
on the phenyl ring) showed a structure similar to tyrosine. The
crucial role of the hydroxyl group at 4-position could be deduced
observing the tyrosinase inhibitory activity of compound 40, char-
acterized by two methoxy substituents at 3-position and 4-position
on the phenyl ring that showed a lower activity (around 10% of
inhibition at 20 mM). At the same time analog 41 (2 methoxy sub-
stituents at positions 2 and 4) exhibited the greatest inhibition of
the l-DOPA oxidase activity of mushroom tyrosinase with an IC50
of 5.05 μM which was 5.31-time fold lower than resveratrol used
as reference compound. Docking simulation investigation pre-
dicted that compound 41 interacts with residue Ser254, Glu258,
and Asp262 in the active pocket through formation of the hydro-
gen and/or ionic bonding. Moreover, kinetic analysis indicates that
this compound acted as a competitive inhibitor of mushroom ty-
rosinase. p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and
p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthe-
sized and evaluated for their inhibition activities on mushroom ty-
rosinase. Results evidenced that both compounds exhibited strong
inhibitory monophenolase activity of the enzyme with IC50 val-
ues of 0.76 and 7.0 μM, respectively. Moreover, HBT and MBT
exhibited potent inhibitory effect against diphenolase activity in a
dose-dependent manner with IC50 values of 3.80 and 2.62 μM,
respectively. Kinetic analyses by Lineweaver-Burk double recip-
rocal plots evidenced that both compounds acted as mixed-type
inhibitors and reversible (Chen and others 2012).
The thiosemicarbazone (Z)-2-(naphthalen-1-ylmethylene)
hydrazinecarbothioamide showed the strongest inhibitory activ-
ity (IC50 value of 1.1 μM) (Lee and others 2010). SAR analysis
evidenced that the position of hydrophobic substituents on the

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phenyl ring of benzaldehydethiosemicarbazones enhances the in-
hibitory activity. Furthermore, the aromatic group of benzalde-
hyde thiosemicarbazones can be replaced with the sterically bulky
cyclohexyl group. Thus, hydrophobicity of the aryl or alkyl group
on hydrazine of thiosemicarbazones is the determinant factor for
their inhibitory activity rather than planarity. Following this obser-
vation, Yi and others (2011) modified thiosemicarbazone pharma-
cophore with the aim to optimize the structure and to increase its
bioactivity. All obtained compounds exhibited dose-dependent in-
hibitory activity on the diphenolase of mushroom tyrosinase. The
compound 1-benzylidenethiosemicarbazone exhibited a stronger
inhibitory activity than kojic acid which was used as reference
(9 μM instead of 102 μM). SAR analysis pointed out that the
addition of a methylene group did slightly enhance the inhibitory
activity, whereas the introduction of an ethylene group led to
drastical bioactivity reduction. A critical aspect for the bioactivity
is the distance that seperates the thiosemicarbazone group from
the aromatic ring. In fact the introduction of a 2-pyridyl sub-
stituent, instead of the phenyl group, produces a much less active
analog. On the contrary, the introduction of a 2-pyrolidinyl or
2-furanyl substituent slightly increased the bioactivity. Using the
phenylmethylenethiosemicarbazones skeleton as a starting point,
Yi and others (2009) synthetized and tested a series of hydroxy-
or methoxy-substituted phenylmethylenethiosemicarbazones. All
tested compounds exhibited an interesting inhibitory activity
against mushroom tyrosinase using l-DOPA as substrate. Among
them compounds 42 (IC50 value of 0.38 μM), 43 (IC50 value of
0.28 μM), 44 (IC50 value of 0.33 μM), and 45 (IC50 value of
0.18 μM) were the most active. In phenylmethylenethiosemicar-
bazones series, compound 46, characterized by a phenyl ring with-
out any type of substituents, exhibited a strong inhibitory activity
(IC50 value of 1.93 μM). In contrast, compounds characterized
by the presence of hydroxyl groups on 2-position and 4-position
of the phenyl ring showed a lower bioactivity. This bioactivity was
drastically reduced if the substituent is in 3-position. A decrease
in inhibitor potency was observed also when the hydroxyl group
in 4-position was replaced with methoxy group. On the contrary
the introduction of a bromine substituent increase the bioactivity.
Compound 45 exhibited the strongest bioactivity probably due
to the 2,4-resorcinol and thiosemicarbazide moieties in its struc-
ture. Authors hypothesized that the presence of an hydrophobic
subunit connected to the resorcinol aromatic ring could help 45
to bind residues His190 and Ala202 located in the hydrophobic
pocket of the enzyme. When the hydroxyl groups were replaced
by methoxy substituents a drastical reduction in the inhibitory ac-
tivity was observed. Moreover, disubstituted compounds showed
stronger inhibitory activity than trisubstituted compounds indicat-
ing that the third substituent might hinder the correct docking of
the inhibitor to the active site of the enzyme. SAR analysis revealed
also that the replacement of benzene moiety of 46 with furan
group as in 2-(2-furanylmethylene)-thiosemicarbazone (47) (IC50
value of 0.45 μM) determined a high potency suggesting that
the electron-rich aromatic ring might be more favorable for the
interaction between the active site of the enzyme and the inhibitor.
In continuing searching of novel tyrosinase inhibitors Yi and
others (2010a) synthesized and tested a series of 4-O-substituted
phenylmethylenethiosemicarbazones. The (1-(1-(4-(2-(2-
methoxyethoxyl)ethoxyl))benzyliene)thiosemicarbazide) (48)
exhibited a strong inhibitory activity with an IC50 value of
0.34 μM. SAR analysis revealed that changement in the number
of oxygen atom and the length of alkyl group could be reduced
the bioactivity of 48 probably due the steric hindrance that
Tyrosinase inhibitors: an update. . .

R
O cyclohexyl S
S R1 R1 H S
N
HO R2
35 OH
O N
36 R= H, R1= OH H
37 R= R1= OH R3
S R4
R2 C N NH C NH2
H
38 R1= R4= H, R2= OH, R3= OMe
R1 39 R1= H, R2=R4= OMe, R3= OH
S
40 R1= R4= H, R2= OMe, R3= OH
42 R1= OH, R2= H C N NH C NH2 41 R1= OMe, R2=R4= H, R3= OMe
43 R1= H, R2=Br O H
44 R1= OH, R2= Br 47
45 R1=R2= OH
46 R1=R2= H S
CH3O(CH2)2O(CH2)2O C N NH C NH2
H

48

Figure 5–Structures of compounds 35–48.

impede the interaction between inhibitor and the active site of
the enzyme. The elongation of alkyl chain determined the loss
of the inhibition further supporting the evidence that the chain
attached on 4-position of phenylmethylenethiosemicarbazone
might play a crucial role in the inhibitory activity (Figure 5).
Moreover, the introduction of secondary alkyl group in the
side chain could be favorable for the enzyme inhibition and the
presence of the –CH2 CH=CH2 group in the side chain was better
than those the introduction of –CH(CH3 )2 group, this is probably
due the possible interaction between the –CH2 CH=CH2 group
with the hydrophobic site of the eznyme. The introduction of a
benzoxyl group had no influence in terms of reduction the activity
while the introduction of a pyridine methyleneoxyl group to 4-
position of 2-(phenylmethylene)-thiosemicarbazone determined
an increase in the IC50 value. Previous investigation demonstrated
that 1-(1-arylethylidene)thiosemicarbazide derivatives showed
strong inhibitory activities against mushroom tyrosinase (Liu
and others 2008). A new series of alkylidenethiosemicarbazide
compounds was synthesized and tested against the diphenolase
activity of mushroom tyrosinase. In particular, 1-(propan-2-
ylidene)thiosemicarbazide (49) (IC50 value of 0.086 μM),
1-propylidenethiosemicarbazide (IC50 value of 0.20 μM),
1-ethylidenethiosemicarbazide (IC50 value of 0.23 μM), and
1-(butan-2-ylidene)thiosemicarbazide (IC50 value of 0.28 μM)
showed strongest inhibitory activity. SAR analysis demonstrated
that the increase of the length of alkyl chain determined a decrease
in the activity probably due the stereo hindrance that prevents
the binding with the catalytic site of the enzyme. Another
important structure requirement is the saturated substituents since
compounds with unsaturated substituents are weaker inhibitors.
A decrease in inhibitory activity was observed also in derivatives
with a carbonyl group corresponding to thiosemicarbazides
without a carbonyl group (Liu and others 2009).
Yi and others (2010b) tested different 4-hydroxybenzaldehyde
derivatives as tyrosinase inhibitors and found the IC50 values
ranged from 0.059 to 3.26 mM. Among the synthetized com-
pounds, the analog with a dimethoxyl phosphate substituent was
the most potent inhibitor. Replacement of a methoxy group with
an ethoxy group drastically reduced the tyrosinase inhibitory activ-
ity, probably due to the dimension of this substituent that hinders
the binding of the compound with the enzyme. The substitution
of the methoxy and methoxyethoxy groups at 4-position of the
phenyl ring led to 2 new tyrosinase inhibitors with IC50 values of
0.66 and 0.9 mM. Moreover, the terminal methoxy group con-
tributed to tyrosinase inhibitory activity, but the elongation of
alkyl chain makes the binding with active site of the enzyme more
difficult. The compound characterized by ethylene oxide moiety
in the side chain showed potent tyrosinase inhibitory activities,
whereas its opened-ring congener exhibited a weaker inhibitory
effect. Summarizing such evidence applied to all synthetized struc-
tures, SAR analysis pointed out that: (1) the numbers of oxygen
atoms contained in the chain substituted at 4-position of benzalde-
hyde modified the enzyme bioactivity; (2) the elongation of the
alkyl chain might retard the binding of inhibitors with the active
site of tyrosinase; (3) the molecular symmetry significantly influ-
enced the enzyme activities; and (4) the introduction of l-glycine,
l-alanine, or l-tryptophan enhanced the enzyme inhibitory ac-
tivities. Another research group synthesized, and evaluated for its
tyrosinase inhibitory activity, a series of bis-salicylaldehydes (De-
logu and others 2010). 5,5 -Methylene-bis-salicylaldehyde (50)
exerted the highest activity (IC50 value of 0.074 mM) which was
10-fold higher than for benzaldehyde. SAR analysis pointed out
that the introduction of a methoxy group in the para position of
compound 50 reduced its bioactivity (IC50 value of 0.117 mM)
while the introduction of an hydroxy group in the same position
led to good inhibitory activity (IC50 value of 0.076 mM) similar
to 50.
Pan and others (2011) synthesized and identified as ty-
rosinase inhibitors three 3,4-dihydroxybenzoates. The 3,4-
dihydroxybenzoate exhibited the strongest inhibitory activity with

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Tyrosinase inhibitors: an update. . .
an IC50 value of 81 μM followed by octyl 3,4-dihydroxybenzoate
and heptyl 3,4-dihydroxybenzoate (IC50 values of 129 and
316 μM, respectively). The bioactivity was potentiated in-
creasing length of hydrocarbon chains. Determination of the
inhibitory mechanism of heptyl 3,4-dihydroxybenzoate and
3,4-dihydroxybenzoate revealed that the 1st was reversible
inhibitor while the 2nd was an irreversible inhibitor. In particular,
the mechanish of inhibition of octyl 3,4-dihydroxybenzoate
depended on the concentration since it was observed that when
the concentration was lower than 50 μM, it showed a reversible
inhibitory mechanism that became irreversible around 75 μM, so
octyl 3,4-dihydroxybenzoate is a mixed-type inhibitor. Analysis
of the double-reciprocal plots indicated that compounds are
uncompetitive inhibitors on the enzyme that means that 3,4-
dihydroxybenzoates could only bind the tyrosinase in the form of
enzyme-substrate complex and not in the free form. Phlorizin is a
flavonoid distributed in fruitss such as apples and pears. Its analogs
were synthesized and tested against mushroom tyrosinase (Fang
and others 2011). 4-Hydroxybenzyl 3,5-dihydroxybenzoate,
4-hydroxybenzyl 2,4-dihydroxybenzoate, 4-hydroxybenzyl 2,4,6-
dihydroxybenzoate, 3-hydroxybenzyl 3,5-dihydroxybenzoate,
and 3-hydroxybenzyl 2,4-dihydroxybenzoate exhibited inhibitory
activity with IC50 less than 10 μM. In particular, 4-hydroxybenzyl
2,4-dihydroxybenzoate exhibited the strongest avctivity with an
IC50 value of 4.95 μM. These 5 compounds were competitive
inhibitors of tyrosinase and the SAR clearly showed that the
position of hydroxyl substituted on ring B remarkably affected
the inhibition. The effect of a series of 4-(phenylurenyl)chalcone
derivatives against banana tyrosinase using cathecol as substrate
were investigated by Sonmez and others (2011). The result
showed that all tested compounds acted in a competitive-manner
as revealed by Lineweaver-Burk double reciprocal plots, with IC50
values ranging from 0.133 to 0.289 μM. Matos and others (2011)
modified the 3-phenylcoumarin scaffold introducing methoxyl,
ethoxyl, hydroxyl, and/or bromo at the 6, 8, and 4 positions as
substituents with the aim to find out any structural features for
the tyrosinase inhibitory activity. The compound characterized
by a bromo atom and 2 hydroxyl groups in the 3-phenylcoumarin
moiety showed the highest potency (IC50 value of 215 mM). The
inhibitory mechanism of this compound was determined using
a Lineweaver-Burk double reciprocal plot. With an increase in
compound concentration, the Vmax value decreased, whereas the
K m value was unchanged, suggesting that this compound is a
noncompetitive tyrosinase inhibitor. SAR analysis revealed that in
the phenylcoumarin series the methoxyl and ethoxyl substitutions
reduced the activity against the enzyme. Seo and others (2010)
reported a series of chalcone derivatives as tyrosinase inhitors.
Among them, 4 ,4-dihydroxychalcone (51) and 4 -amino-4-
hydroxychalcone (52) showed IC50 values of 4.8 and 8.3 mM,
respectively. Kinetics analysis by Lineweare-Burk plot indicated
that all compounds acted as competitive inhibitors. Critical for
the srong bioactivity of all synthtized chalcones apparently is the
presence of sterically unencumbering groups on the A ring.
Curcumin is a major ingredient of turmeric, a spice powder
obtained from the rhizome of Curcuma longa. Du and others
(2011) isolated and tested curcumin, demethylcurcumin, and
bis-demethylcurcumin against mushroom tyrosinase finding IC50
values of 94.73, 53.03, and 33.50 μM. Using these natural
curcuminoids, the authors designed several analogs possessing
m-diphenols and o-diphenols. All tested compounds resulted in
more activity than kojic acid used as reference compound. In
particular, 1,5-bis-(2,4-dihydroxybenzylidene)penta-1,4–3-one

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(53) member of m-diphenol group exhibited the strongest
inhibitory activity with an IC50 value of 0.65 μM. When a
methoxy group replaced an hydroxyl group in 4-position the
bioactivity decreased. The crucial role of 4-position is clearly
visible. Moreover, the order of bioactivity of compounds depend
on the linkers so compounds with acetone linkers > compounds
with cyclopentanone > compounds with tetrahydropyran-4-
one > compounds with cyclohexanone > compounds with
tetrahydrothiopyran-4-one > piperidin-4-one. The inhibition
kinetics, analyzed by Lineweaver–Burk plot, revealed that com-
pounds with o-diphenol acted as noncompetitive inhibitors, while
compounds m-diphenols could be bound only by the free form
of the enzyme. Data regard the kinetics are controversial since
previously described compounds characterized by m-diphenols
showed competitive inhibitory activity (Song and others 2006;
Baek and others 2009). Molecular docking study revealed that
both 2,5-bis-(3,4-dihydroxybenzylidene)cyclopentanone (54)
and 2,5-bis-(2,4-dihydroxybenzylidene)cyclopentanone (55)
formed a π-π link between one aromatic group and His194
localized in the active pocket of the enzyme together with
multiple hydrogen bonds that involved the hydroxylic groups.
In particular, o-diphenol compounds such as compound 54
formed four hydrogen bond with Gly183, Trp184, Ser206 while
m-diphenol compounds such as compound 55 formed seven
hydrogen bond with Ser206, Ala202, Asn191, Asn188, Thr203,
and Ser146 residues. The number of hydrogen bonds with the
active site of the enzyme could explain the stronger inhibitory
activity of m-diphenol compounds respect the o-diphenol com-
pounds. Successively, Hosoya and others (2012) synthetized 47
curcumin-like diarylpentanoid analogues as potential tyrosinase
inhibitors. Among them (1E,4E)-1,5-bis(4-hydroxyphenyl)penta-
1,4-dien-3-one exhibited an IC50 value of 6.4 μM. Compound
(1E,4E)-1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-
one, that possesses as curcumin a methoxy group at 3-position
and an hydroxyl group at 4-position, showed an higher inhibitory
activity (IC50 value of 75.5 μM) than its parent compound
curcumin. In agreement with Du and others (2011), SAR
analysis revealed that the substitution of an hydroxyl group at the
4-position is crucial for a strong inhibitory activity (Figure 6).
Choi and others (2010) synthesized a derivative of resvera-
trol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (56), which
showed potent tyrosinase inhibitory activity (IC50 value of
2.95 μM). Kinetics analysis by the Lineweare–Burk plot indicated
that compound 56 acts as noncompetitive inhibitor when
l-tyrosine was used as substrate.
From pinapple, the 2 sulfur-containing compounds N-l-
γ -glutamyl-S-sinapyl-l-cysteine and S-sinapyl-l-cysteine were
isolated and tested for tyrosinase inhibitory activity, finding IC50
values of 237.33 and 199.04 μM, respectively (Zheng and others
2010b).
A series of bibenzyl glycosides was synthesized and evalu-
ated for their tyrosinase inhibitory activity. All of the synthe-
sized bibenzyl glycosides exhibited strong inhibitory activities
(Tajima and others 2011). In particular, 2,2 ,4 -trihydroxy-4-β-d-
xylopyranosylbibenzyl (57), characterized by bulky bibenzyl gly-
cosides, exhibited an IC50 value of 0.43 μM that was 17 times
higher than that of kojic acid used as reference. Chemical charac-
teristics of compound 57 suggest that the enzyme might possess
a hydrophilic cavity at its catalytic site. Takahashi and Miyazawa
(2011) synthesized a series of phenylpropanoid amides of sero-
tonin and analyzed their ability to inhibit the tyrosinase. Among
the tested derivatives, N-Isoferuloyl serotonin (58) showed the
Tyrosinase inhibitors: an update. . .
S
N NH C NH2 OHC
C
H R
49 HO
50
O
R3 R1 R1 R3
R2 R2
53 R2= H, R1= R3= OH
54 R1= H, R2= R3= OH
Figure 6–Structures of compounds 49–55.
most promising inhibitory activity (IC50 of 5.4 μM). Lineweaver-
Burk plot revealed that compound 58 acted as a noncompeti-
tive inhibitor of mushroom tyrosinase. Previously, Bao and others
(2010) synthetized a series of natural product biphenyl glyco-
side fortuneanoside E analogs. Among them, 3,4 -dihydroxy-3 ,5 -
dimethoxybiphenyl-4-carboxylic acid (59) exhibited the highest
bioactivity with an IC50 value of 0.02 mM acting as competitive
inhibitor (Ki = 0.015 mM). This activity was 7 times higher than
that of fortuneanoside E (IC50 value of 0.14 mM) and 10 times
higher than that of arbutin a known as potent tyrosinase inhibitors.
SAR analysis showed that the presence of a 4-hydroxy-3,5-
dimethoxyphenyl moiety was critical for the inhibitory activity.
Several 5-(substituted benzylidene)hydantoin derivatives were
synthesized and tested against mushroom tyrosinase enzyme (Ha
and others 2011). In terms of the SAR analysis, compound 60,
characterized by 2 hydroxy groups on the phenyl ring despite
having a structure similar to DOPA, did not inhibit mushroom
tyrosinase. Furthermore, compound 61, with a hydroxyl group
on the phenyl ring, showed low activity despite having a structure
similar to tyrosine. Compound 62, characterized by a phenyl ring
substituted with a hydroxyl group and a methoxy group showed
the highest inhibitory activity. Compound 63, with these groups
inverted and compound 64 with 2 methoxy groups, demonstrated
poor inhibitory activity. A docking study brought evidence that
compound 62 interacts strongly with the enzyme as a competitive
tyrosinase inhibitor.
Several SAR studies have identified hydroxyl as the functional
group required for tyrosinase inhibitory activity (Yokota and
others 1998; Shiino and others 2001, 2003; Kim and others 2006).
The hydroxyl groups in compounds carry out the nucleophilic
attack on the copper atoms in the active site of the enzyme and are
directly involved in transferring protons during catalysis, which
then result in inactivation of the tyrosinase. In this context, using
inhibition kinetics and computational simulation the reversible
inhibition of tyrosinase by isophthalic acid was studied (Si and
others 2011). Isophthalic acid inhibited tyrosinase in a complex
manner with K i of 17.8 mM, without modification of the
tertiary protein structure. In this study, the authors predicted that
isophthalic acid could bind to the Pro175 or Val190.
Some of the synthesized compounds of a series of
dihydropyrimidin-(2H)-one analogs and rhodanine deriva-
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O
CHO
OH
OH
51 R= NH2
52 R= OH
O
HO OH HO OH
55
tives exhibited significant inhibitory tyrosinase activity (Liu
and others 2011). Rhodanine derivatives inhibited the enzyme
more than the dihydropyrimidin-(2H)-one analogues, which
indicated that the smaller size of the ring could help the molecules
to interact with the active site of the enzyme. Among this
series compound 65 characterized by a hydroxyethoxyl group
at position-4 of the phenyl ring, exhibited the most potent
inhibitory activity (IC50 value of 0.56 mM). The kinetics analysis
demonstrated that the inhibitory effect of compound 65 was
irreversible. Comparing it to the enzyme inhibitory activities
of 3,4-dihydropyrimidin-2-(1H)-thione analogs, as previously
hypothesized compounds with a hydroxyl group in the benzene
ring showed stronger inhibitory activities probably due the ability
of hydroxyl groups to create a metal-ligand binding interaction
with the dicopper nucleus (Ghani and Ullah 2010). Moreover, the
tyrosynase inhibitory activity was higher when the hydroxyl group
was introduced in the p-position with respect to the o-position.
If the substituent is introduced in the m-position on the phenyl
rings a decrease in inhibitory activities is observed (Figure 7).
Eight vic-substituted o-aminophenols belonging to 2 isomeric
series were investigated for their tyrosinase inhibitory activity by
Rescigno and others (2011). Four o-aminophenolic compounds
derived from the 3-hydroxyorthanilic acid scaffold and their 4
counterparts derived from the isomeric 2-hydroxymethanilic acid
were investigated as mushroom tyrosinase inhibitors. Among the
synthetized compounds, hydroxymethanilic compounds acted as
substrates for the enzyme, which oxidized them to the correspond-
ing phenoxazinone derivatives. Analysis of the results revealed
that aromatic amines, o-aminophenols, and even o-diamines could
represent tyrosinase substrates. In fact, amines are hydroxylated
to the corresponding o-aminophenols, and o-aminophenols
are oxidized to the corresponding o-quinoneimines. A similar
behavior was observed also with o-diamines, o-aminophenols,
and o-diphenols. On the contrary, the substitution of nitrogen for
oxygen caused a drastic reduction in the reaction speed.
Arbutin (4-hydroxylphenyl-O-β-d-glucopyranoside) is a well-
known tyrosinase inhibitor. Yi and others (2008) designed,
synthesized, and tested a series of helicid (4-formylphenyl-O-β-
d-allopyranoside) derivatives. These compounds are characterized
by analogy with arbutin’s chemical structure. 4-Formylphenyl
(4,6-O-benzylidene)-β-d-allopyranoside (66) demonstrated the

c 2012 Institute of Food Technologists®
Tyrosinase inhibitors: an update. . .

H
OH N
OH O
OH
HO
N
H
HO OH MeO OH
56 XylO 57
OH OH 58

O O
R NH
OMe
NH
OH HN S S
R1
O
HO
OMe 60 R= R1= OH
61 R= H, R1= OH
HOOC 62 R= OH, R1= OMe
59 OCH2CH2OH
63 R= OMe, R1= OH
64 R= OMe, R1= OMe 65

Figure 7. Structures of compounds 56–65.

OAc

AcO O
Ph O S 67 R=
O O AcO
OAc
N
H2N HN OR OAc OAc
HO OH O CHO
AcO O O
68 R= O
66 AcO
OAc AcO OAc

OH
OH
OH OH
O O OH
O O O O OH
HO O
OH HO O
69 OH
70

Figure 8–Structures of compounds 66–70.

strongest enzyme inhibitory activity (IC50 value of 0.052 mM).
SAR analysis indicated that the presence of 4,6-O-benzylidene,
2,3-O-isopropylidene, and 6-O-dimethyl phosphate into sugar
moiety could help the interaction with the enzyme probably
due the lipophilic character of these substituents. Moreover, a
β-d-glucopyranoside moiety was better than β-d-allopyranoside
moiety in the enzyme inhibitory activity. In regards to the type
of sugar it is clear that the ribose moiety was more preferable
than glucose moiety. Kinetics analysis showed that helicid and
analogues were competitive inhibitors of the mushroom tryrosi-
nase while the circular dichroism spectra indicated that those
compounds induced conformational changes of mushroom ty-
rosinase upon binding. Based on the evidence that the modifica-
tion of glycosyl moiety of arbutin might facilitate the inhibitory
activity against tyrosinase, the same research group designed, syn-
thesized and evaluated a series of novel 4-functionalized phenyl-
O-β-d-glycosides using thiosemicarbazide, oxime, and methy-
loxime skeleton (Yi and others 2009). SAR analysis revealed that
compounds characterized by thiosemicarbazide moiety demon-
strated significant antityrosinase activity. In particular, p-phenyl-
tetra-O-acetyl-β-d-glucopyranoside benzaldehyde (67) showed
an IC50 value of 0.31 μM. The presence of β-d-allopyranoside or
β-d-glucopyranoside moiety did not influenced the activity. On
the contrary the presence of tetra-O-acetyl-β-d-glucopyranoside
moiety was favorable for the enzyme interaction. Interestingly,
the replacement of this sugar moiety with tetra-O-acetyl-β-d-
galactopyranoside moiety did not affect the bioactivity. At the same
time the replacement of acetyl substituents with benzoyl groups
determinated a drastically reduction in the inhibitory potency. In
order to investigate if the volume of the sugar moiety could be
influenced, the enzyme inhibitory activity of hepta-O-acetyl-β-
d-lactoside analog was designed and tested, finding an IC50 value


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Tyrosinase inhibitors: an update. . .

Table 2–Isolated natural compounds with tyrosinase inhibitory activity.

Compounds IC50 Type of inhibition

Kuwanon C 49.2 μM Competitive
Sanggenon D 7.3 μM Competitive
Rosmarinic acid 16.8 μM Competitive
Rosmarinic acid methyl ester 21.5 μM Competitive
Pedalitin 0.28 mM Mixed-type
Silybin A 21 μM NR
Isosilybin A 10 μM NR
Isosilybin B 6.1 μM NR
Luteolin 14 μM NR
Kaempferol 25 μM NR
Quercetin 15 μM NR
Hesperetin 11.25 mM Competitive
Hierochin A 25 μM NR
(+)-Dehydrodiconiferyl alcohol 16 μM NR
(+)-Balanophonin 15 μM NR
Hierochin B 30 μM NR
3,4-Dihydroxybenzaldehyde 17 μM NR
Arbutin 174 μM NR
N-p-Coumaroylserotonin 0.027 mM NR
N-Feruloylserotonin 0.027 mM NR
3-O-p-Coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone 0.055 mM NR
3-O-p-Coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-O-β-glucopyranosylpropanol 0.053 mM NR
Ethyl-α-d-glucopyranoside 0.19 mg/mL NR
Adenosine 0.13 mg/mL NR
Barbarin 4.2 × 10−5 M NR
Epigallocatechin 8 mM Competitive
Epigallocatechin gallate 0.7 mM Competitive
Gallocatechin 4.8 μg/mL NR
epi-Gallocatechin gallate 30.2 μg/mL NR
Methyl gallate 40.5 μg/mL NR
Quercitrin 37.3 μg/mL NR
p-Octyl benzoic acid 0.047 mM Noncompetitive
p-Propyl benzoic acid 0.213 mM Noncompetitive
p-Hydroxybenzoic acid 0.98 mM Competitive
Methyl p-hydroxybenzoate 0.66 mM Competitive
3,5,7,4 -Tetrahydroxy-30-(2-hydroxy-3-methylbut-3-enyl)flavones 96.6 μM NR
Uralenol 49.5 μM NR
Broussoflavonol F 82.3 μM NR
Artocarpfuranol 47.93 μM NR
Dihydromorin 10.3 μM NR
Steppogenin 0.57 μM NR
Norartocarpetin 0.46 μM NR
Artocarpanone 1.54 μM NR
Artocarpesin 0.52 μM NR
Isoartocarpesin 0.66 μM NR
(Z,Z)-5-(Trideca-4,7-dienyl)-resorcinol 0.449 μg/mL NR
Dalenin 0.26 μM Noncompetitive
Sophoraflavanone G 4.7 μM Noncompetitive
Kurarinone 2.2 μM Noncompetitive
Kurarinol 0.1 μM Noncompetitive
5 -Geranyl-5,7,2 ,4 -tetrahydroxyflavone 37.09 μM NR
2,4,2 ,4 -Tetrahydroxychalcone 0.062 μM NR
Morachalcone A 0.14 μM NR
Kuwanon E 77.9 μM NR
Moracin C 111.47 μM NR
Moracin N 30.52 μM NR
2 ,4 ,6 -trihydroxydihydrochalcone 69.15 μM NR
Cardol triene 22.5 μM Competitive
2,3-cis-Dihydromorin 31.1 μM NR
2,3-trans-Dihydromorin 21.1 μM NR
Oxyresveratrol 2.33 μM NR
Quercetin-7-O-β-d-glucoside 143.07 μM NR
Kaempferol-7-O-β glucopyranoside 161.54 μM NR
Morin-7-O-β-d-glucoside 196.33 μM NR
Quercetin-3,7-di-O-β-d-glucoside > 1000 μM NR
6,7,4-Trihydroxyisoflavone 0.009 mM Competitive
Genistin 0.343 mM Competitive
Daidzein 0.203 mM Competitive
Glycitein 0.218 mM Competitive
Daidzin 0.267 mM Competitive
Vitexin 6.3 mg/mL NR
Isovitexin 5.6 mg/mL NR
Lariciresinol 4-O-β-d-glucoside 42.59 μM NR
Threo-guaiacylglycerol-7-O-β-d-glucopyranoside 57.72 μM NR
Erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol-4 -O-β-d-glucopyranoside 85.87 μM NR
Threo-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol 4 -O-β-d-glucopyranoside 95.81 μM NR
2-(3-methoxyl-4-hydroxyphenyl)-3-Hydroxylmethyl-5-trans-carboxylethylene-7-methoxyl-2,3- 798.02 μM NR
dihydrobenzofuran

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Tyrosinase inhibitors: an update. . .
Table 2–Continued.
Compounds
Arabinose
Dieckol
Curcumin
Demethylcurcumin
bis-Demethylcurcumin
Chrysontemin
Mimosine
Helicid
Costinones A
Costinones B
Isatinones A
Isatinones B
Indirubin
Trisindoline
NR: not reported.
of 0.65 μM p-phenyl-hepta-O-acetyl-β-d-lactoside benzalde-
hyde (68). Collectively all these observations revealed that the
configuration and bond type of sugar moiety played a very
important role in the enzyme inhibitory activity and that the
lipophilic character of acetylated sugar moiety could increase the
potency of the inhibitors. Both 67 and 68 demonstrated to act
as reversible inhibitors. This finding is in disagreement with the
mode of action of 1-phenylthiourea and 1-(1-phenylethylidene)
thiosemicarbazide analogs that acted as irreversible inhibitors. Par-
ticularly, the Lineweaver-Burk double reciprocal plots showed that
67 was a competitive inhibitor of mushroom tyrosinase whereas
68 could be considered as mixed-II-type inhibitor. The kinetics
behavior of 67 lead the authors to hypothesize that compound 67
could interact with the enzyme through two different portions
of its structure: the sulfur atom of the thiosemicarbazide moiety
chelate, the binuclear copper of the enzyme, and such interaction
acted as a bridge to link the acetylated glucose moiety and the hy-
drophobic protein pocket, which facilitated the acetylated glucose
moiety to approach the hydrophobic protein pocket. Whereas
the large size of lactoside moiety that characterized compound 68
prevented by binding to the active site of the complex enzyme-l-
DOPA.
5-Benzylidene barbiturate and thiobarbiturate derivatives were
synthetized and tested by Yan and others (2009). These com-
pounds displayed more potent tyrosinase inhibitory activities than
that parent compounds barbituric acid, thiobarbituric acid, and
4-hydroxybenzylaldehyde. In the most active series compounds 5-
(4-(2-methoxyethoxy)benzylidene)-2-thioxodihydropyrimidine-
4,6(1H,5H)-dione, and 5-(4-(2-(2-methoxyethoxy)ethoxy)
benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione dis-
played interesting IC50 values of 28.43 and 34.20 μM, respectively.
SAR analysis clearly revealed that the thiobarbiturate moiety was
more favorable than barbiturate moiety in inhibiting the dipheno-
lase activity of mushroom tyrosinase. As previously evidenced in
different types of compounds an hydroxyl group at 4-position of
phenyl ring determined a strong inhibitory activity as compounds
5-(4-hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione
and 5-(4-hydroxybenzylidene)-2-thioxo-dihydropyrimidine-4,6
(1H,5H)-dione with IC50 values of 13.98 and 14.49 μM, respec-
tively. According to previous investigation the replacement of the
hydroxyl group with alkyl chain drastically reduced the bioactivity
and this reduction is consequent also to the length of alkyl chain
since it causes a steric hindrance that prevents the binding with
the catalytic site of the enzyme. Kinetics studies revealed that
the most potent inhibitor 5-(4-hydroxybenzylidene)pyrimidine-

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IC50 Type of inhibition
0.1 mM Mixed-type
20 μM Noncompetitive
94.73 μM NR
53.03 μM NR
33.50 μM NR
211 μM Competitive
3.70 μM Mixed-type
2.54 mM Competitive
7.21 μM NR
9.40 μM NR
11.51 μM NR
12.53 μM NR
14.29 μM NR
17.34 μM NR
2,4,6(1H,3H,5H)-trione and 5-(4-hydroxybenzylidene)-2-
thioxo-dihydropyrimidine-4,6(1H,5H)-dione acted as irreversible
inhibitors. Moreover, the circular dichroism spectra showed that
both compounds induced conformational changes of the enzyme
(Yan and others 2009).
A series of caffeoyl-amino acidyl-hydroxamic acid derivatives
showed an interesting enzyme inhibitory activity compared to
their parent compound In particular, CA-L/d-Phe-NHOH,
CA-Phg-NHOH, CA-Leu-NHOH, CA-Ala-NHOH, and CA-
Pro-NHOH exhibited higher inhibitory activity than derivatives
CA-β-Ala-NHOH and CA-Gly-NHOH. This different behavior
could be explained considering that different character of amino
acids inserted in derivatives structure. In particular, hydrophobic
alkyl chain of Leu and or the aromatic ring of Phe could make
easier the chelation of dinuclear copper located on the active site
of the enzyme by hydroxamic acid. Interestingly, CA-Pro-NHOH
has hydrophobic character that allows to permit the binding to
the active site of the enzyme (Kwak and others 2011).
The mushroom tyrosinase inhibitory activity of 2-
phenylethanol, 2-phenylacetaldehyde, and 2-phenylacetic acid has
recently been investigated (Zhu and others 2011). All compounds
acted as reversible inhibitors; furthermore, 2-phenylacetaldehyde
and 2-phenylacetic acid were shown to be uncompetitive
inhibitors and 2-phenylethanol a mixed-type inhibitor. The
inhibiting ability was influenced by the position of the functional
group on the benzene ring, in particular 2-phenylacetaldehyde
which inhibited the enzyme more than 2-phenylacetic acid or
2-phenylethanol.
Recently, Nesterov and others (2008) synthetized and tested
the natural product 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-
3-methylphenyl) propane isolated for the 1st time in Dianella
ensifolia. This compound was 22 times more potent than kojic
acid for inhibiting mushroom tyrosinase activity in a competitive
and reversible fashion (Ki = 0.3 μM).
It is well known that vitamin C has potent tyrosinase inhibitory
activity and is largely used in the food industry for this purpose.
Based on this consideration Yi and others (2009) reported the
strong inhibitory activity of vitamin C esters namely d-ascorbic
acid-6-p-hydroxybenzoic acid ester (69, IC50 value of 0.58 mM)
and d-ascorbic acid-6-(3,4,5-hydroxybenzoic acid) ester (70,
IC50 value of 0.16 mM) on the diphenolase activity of mushroom
tyrosinase. The kinetic analysis revealed that the 2 esters behaved
differently since compound 70 was a reversible inhibitor and its
mechanism was mixed type and ester 69 was an irreversible
inhibitor (Figure 8 and Tables 2 and 3).
Tyrosinase inhibitors: an update. . .

Table 3–Synthetic compounds with tyrosinase inhibitory activity.

Compounds IC50 Type of inhibition

Cinnamic acid 2.10 mM Noncompetitive
4-Hydroxycinnamic acid 0.5 mM Competitive
4-Methoxycinnamic acid 0.42 mM Noncompetitive
α-Cyano-4-hydroxycinnamic acid 0.28 mM Competitive
Rosmarinic acid methyl ester 2.17 mM Competitive
2-(Cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one 0.087 μM NR
2-(Pentylthiomethyl)-5-hydroxy-4H-pyran-4-one 0.097 μM NR
p-Hydroxybenzaldehyde thiosemicarbazone 0.76 μM (monophenolase); Reversible/ Mixed-type
3.8 μM (diphenolase)
p-Methoxybenzaldehyde thiosemicarbazone 7 μM (monophenolase); Reversible/ Mixed-type
2.62 μM (diphenolase)
(Z)-2-(Naphthalen-1-ylmethylene)hydrazinecarbothioamide 1.1 μM NR
1-Benzylidenethiosemicarbazone 1 μM NR
2-[(2-Hydroxyphenyl)methylene]-thiosemicarbazone 0.38 μM NR
2-[(4-Bromophenyl)methylene]-thiosemicarbazone 0.28 μM NR
2-[(2-Hydroxy-4-bromophenyl)methylene]thiosemicarbazone 0.33μM NR
2-[(2,4-Dihydroxyphenyl)methylene]-thiosemicarbazone 0.18 μM NR
2-(Phenylmethylene)-thiosemicarbazone 1.93 μM NR
2-(2-Furanylmethylene)-thiosemicarbazone 0.45 μM NR
(1-(1-(4-(2-(2-Methoxyethoxyl)ethoxyl))benzyliene)thiosemicarbazide) 0.34 μM NR
1-(Propan-2-ylidene)thiosemicarbazide 0.086 μM NR
1-Propylidenethiosemicarbazide 0.20 μM NR
1-Ethylidenethiosemicarbazide 0.23 μM NR
1-(Butan-2-ylidene)thiosemicarbazide 0.28 μM NR
5,5 -Methylene-bis-salicylaldehyde 0.074 mM NR
4,4 -Dimethoxy-5,5 -methylene-bis-salicylaldehyde 0.117 mM NR
4,4 -Dihydroxy-5,5 -methylene-bis-salicylaldehyde 0.076 mM NR
3,4-Dihydroxybenzoate 81 μM Irreversible
octyl 3,4-Dihydroxybenzoate 129 μM Mixed type/Uncompetitive
heptyl 3,4-Dihydroxybenzoate 316 μM Reversible/Uncompetitive
4-Hydroxybenzyl 2,4-dihydroxybenzoate 4.95 μM NR
4 ,4-Dihydroxychalcone 4.8 mM Competitive
4 -Amino-4-hydroxychalcone 8.3 mM Competitive
Curcumin 94.73 μM NR
Demethylcurcumin 53.03 μM NR
bis-Demethylcurcumin 33.50 μM NR
1,5-bis-(2,4-Dihydroxybenzylidene)penta-1,4–3-one 0.65 μM Competitive
2,5-bis-(3,4-Dihydroxybenzylidene)cyclopentanone 1.19 μM Noncompetitive
2,5-bis-(2,4-Dihydroxybenzylidene)cyclopentanone 0.78 μM Competitive
(1E,4E)-1,5-bis(4-Hydroxyphenyl)penta-1,4-dien-3-one 6.4 μM NR
5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol 2.95 μM Noncompetitive
N-L-γ -Glutamyl-S-sinapyl-l-cysteine 237.33 μM NR
S-Sinapyl-l-cysteine 199.04 μM NR
2,2 ,4 -Trihydroxy-4-β-d-xylopyranosylbibenzyl 0.43 μM NR
N-Isoferuloyl serotonin 5.4 μM Noncompetitive
3,4 -Dihydroxy-3 ,5 -dimethoxybiphenyl-4-carboxylic acid 0.02 mM Competitive
Fortuneanoside E 0.14 mM NR
4-Hydroxyethoxyldihydropyrimidin-(2H)-one 0.56 mM Irreversible
4-Formylphenyl (4,6-O-benzylidene)-β-d-allopyranoside 0.052 mM Competitive
p-Phenyl-tetra-O-acetyl-β-d-glucopyranoside benzaldeide 0.31 μM Reversible
p- Phenyl-hepta-O-acetyl-b-d-lactoside benzaldeide 0.65 μM Reversible
5-(4-(2-Methoxyethoxy)benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione 28.43 μM NR
5-(4-(2-(2-Methoxyethoxy)ethoxy)benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione 34.20 μM NR
5-(4-Hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione 13.98 μM Irreversible
5-(4-Hydroxybenzylidene)-2-thioxo-dihydropyrimidine-4,6(1H,5H)-dione 14.49 μM Irreversible
d-Ascorbic acid-6-p-hydroxybenzoic acid ester 0.58 mM Irreversible
d-Ascorbic acid-6-(3,4,5-hydroxybenzoic acid) ester 0.16 mM Mixed-type
NR: not reported.

Concluding Remarks
The object of this review article was to gather and present an
up-to-date display of natural and synthetic compounds able to
inhibit tyrosinase. This enzyme is regarded to play a critical role
during food handling, storage, and commercial or domestic pro-
cessing. In particular, in plant foods it causes undesirable enzymatic
browning, especially in bruised or cut fruits and vegetables, which
subsequently leads to a significant decrease in nutritional and
market values. The information offered in this review should help
to provide leads to the ultimate goal of developing new antityrosi-
nase inhibitors (plant extracts, natural, and synthetic compounds)
satisfactory efficacy, safety, and useful for the food industry.
The extracts from Dalea elegans, Lithrea molleoides, Greyia flana-
ganii, Cudrania cochinchinensis, and Distylium racemosum could be
indicated among the most active tyrosinase inhibitors from natural
sources. All these extracts showed flavonoids as main constituents.
This class of natural occurring compounds is the largest group in
tyrosinase inhibitors. Several studies have been demonstrated that
the number and position of hydroxyl group of the B ring as well as
the substituents with steric hindrance jock a key role in the tyrosi-
nase inhibitory activity. The hydroxyl groups in compounds carry
out the nucleophilic attack on the copper atoms in the active site of
the enzyme and are directly involved in transferring protons during
catalysis, which then result in inactivation of the tyrosinase. Regard

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Tyrosinase inhibitors: an update. . .
the steric hindrance of prenyl group, sugars, or alkyl chain, all these
substituent could be hindering the entry of the inhibitor into the
active pocket of the enzyme. Our data collection clearly evidenced
that steppogenin, artocarpesin, norartocarpetin, artocarpanone,
isoartocarpesin, dalenin, and kurarinol were the most active nat-
ural compounds with IC50 values ranging from 0.1 to 1.54 μM.
Many efforts have been spent in the search for effective
and safe tyrosinase inhibitors, and a large number of synthetic
tyrosinase inhibitors have already been reported. Among them
4-formylphenyl (4,6-O-benzylidene)-β-d-allopyranoside (IC50
value of 0.052 μM), 1-(propan-2-ylidene)thiosemicarbazide (IC50
value of 0.086 μM), 2-(cyclohexylthiomethyl)-5-hydroxy-4H-
pyran-4-one (IC50 value of 0.087 μM), and 2-(pentylthiomethyl)-
5-hydroxy-4H-pyran-4-one (IC50 value of 0.097 μM) exhibited
the highest inhibitory activity. The 4-formylphenyl (4,6-O-
benzylidene)-β-d-allopyranoside is an helicid analog which
presents analogy with a well-known tyrosinase inhibitor arbutin’s
chemical structure (4-hydroxylphenyl-O-β-d-glucopyranoside).
Results evidenced that the modification of glycosyl moiety of
arbutin might facilitate the inhibitory activity against tyrosi-
nase. The 1-(propan-2-ylidene)thiosemicarbazide is a member
of alkylidenethiosemicarbazide series. In thiosemicarbazide
compounds the position of hydrophobic substituents on the
phenyl ring enhances the inhibitory activity. Another critical
aspect is the distance that separates the thiosemicarbazone
group from the aromatic ring. Moreover, important struc-
ture requirements are the presence of saturated substituents.
2-(Cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one and
2-(pentylthiomethyl)-5-hydroxy-4H-pyran-4-one are kojic acid
derivatives. The presence of thioether linkage and lipophilic acid
moiety was critical for the tyrosinase inhibitory activity.
However, more research is needed in investigating if the potent
inhibitors reported in this review article could be introduced
for pratical use and are compatible with the safety regulations in
order to be commercialized as food additives.
Acknowledgment
The authors wish to thank Dr. Anna Internò, Faculty of Phar- berries. Food Sci Biotechnol 18:362–6.
macy, Nutrition and Health Sciences, Univ. of Calabria for the Fang Y, Chen Y, Feng G, Ma L. 2011. Benzyl benzoates: new phlorizin
proof-reader version of the manuscript.
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