Identification of geranic acid, a tyrosinase inhibitor in lemongrass (Cymbopogon citratus)

Article abstract of DOI:10.1021/jf072893l

Lemongrass is a popular Asian herb having a lemon-like flavor. Very recently, potent tyrosinase inhibitory activity has been found in lemongrass in addition to various biological activities reported in the literature. The aim of the present study is to identify the active compounds in the lemongrass. An assay-guided purification revealed that one of the active substances was geranic acid. Geranic acid has two stereoisomers, which are responsible for the trans and cis geometry on the conjugated double bond. Both isomers are present in the active ethyl acetate-soluble extract of the lemongrass, and their IC ₅₀ values were calculated to be 0.14 and 2.3 mM, respectively. The structure requirement of geranic acid for the potent tyrosinase inhibitory activity was investigated using geranic acid-related compounds.

Full text of DOI:10.1021/jf072893l

J. Agric. Food Chem. 2008, 56, 597–601 597
Identification of Geranic Acid, a Tyrosinase Inhibitor
in Lemongrass (Cymbopogon citratus)
TOSHIYA MASUDA,*,# YUKA ODAKA,# NATSUKO OGAWA,#
§
KATSUO NAKAMOTO,§ AND HIDEKI KUNINAGA
Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan, and
Nakazen, Inc., Nanjo, Okinawa 901-1513, Japan
Lemongrass is a popular Asian herb having a lemon-like flavor. Very recently, potent tyrosinase
inhibitory activity has been found in lemongrass in addition to various biological activities reported in
the literature. The aim of the present study is to identify the active compounds in the lemongrass. An
assay-guided purification revealed that one of the active substances was geranic acid. Geranic acid
has two stereoisomers, which are responsible for the trans and cis geometry on the conjugated double
bond. Both isomers are present in the active ethyl acetate-soluble extract of the lemongrass, and
their IC50 values were calculated to be 0.14 and 2.3 mM, respectively. The structure requirement of
geranic acid for the potent tyrosinase inhibitory activity was investigated using geranic acid-related
compounds.
KEYWORDS: Lemongrass; geranic acid; Cymbopogon citratus; tyrosinase inhibition; enzymatic oxidation
INTRODUCTION
in the subtropical Okinawa area of Japan (6). From the
screening, lemongrass was selected as one of the potent active
species.
The oxidative deterioration of food involves two components,
which are nonenzymatic and enzymatic oxidations. Nonenzy-
matic oxidation is observed in all foods. It is induced by light
irradiation, the redox reaction of a transition metal, and
degradation of the oxidized impurity (1). This oxidation can be
protected by antioxidants. Enzymatic oxidation is observed in
fresh foods such as vegetables, fruits, crops, and marine
products. The oxidation proceeds by catalytic action of a food-
bearing oxidation enzyme and makes rapid progress during food
processing (2). This oxidation can be prevented by using a
typical inhibitor for the respective enzyme. The control of both
types of oxidations is very important to preserve the food quality.
Tyrosinase is one of the oxidation enzymes widely distributed
in fresh foods. It catalyzes the oxidation of the phenolic
constituents that results in the browning of food, which reduces
the market value of food. To control the harmful tyrosinase
activity, the development of an efficient inihibitory substance
is required. Many tyrosinase inhibitors have already been
developed by synthesis or isolation from natural sources (3, 4),
because the tyrosinase inhibitor is applicable not only for food
but also for cosmetics such as a skin-whitening agent. Recently,
consumers have shown a preference for natural substances as
safe food ingredients; therefore, various useful agents for food
should be sought in natural resources. We have screened the
tyrosinase inhibitory activity of 53 parts of 36 plant species (5),
which were cultivated for traditionally edible and medicinal uses
Lemongrass (Cymbopogon citratus) is widely used as an
essential ingredient in tropical Asian cuisine due to its lemon-
like flavor (7). In Japan, tea prepared from the dry leaves of
lemongrass is taken for physical and mental health promotion.
Although various bioactivities of lemongrass and their applica-
tion studies have already been reported [antibacterial (8–15),
antifungal (16–18), mosquito repellent (19), insecticide (20),
cytotoxic (21), anti-inflammatory (22), and antioxidant (23)
activities], its potent tyrosinase inhibitory activity has been
newly observed in our studies (5). We report in this paper the
identification of the tyrosinase inhibitor in lemongrass and
structural requirement of the inhibitor for activity presentation.
MATERIALS AND METHODS
Materials and Instruments. Dry leaves of lemongrass (C. citratus)
were purchased from Mikuni Co. Ltd. (Osaka, Japan). Tyrosinase (from
mushroom), L-DOPA (L-3,4-dihydroxyphenylalanine), geranic acid
(purity ) 85%), and activated manganese dioxide were purchased from
Sigma-Aldrich Japan (Tokyo, Japan). Kojic acid, dicyclohexylcarbo-
diimide, and silver oxide were obtained from Wako Pure Chemicals
(Osaka, Japan). Farnesol, 3-methylcrotonic acid, crotonic acid, tiglic
acid, angelic acid, and methacrylic acid were purchased from Tokyo
Kasei (Tokyo, Japan). Silica gel for column chromatography (silica
gel 60 no. 9385) and silica gel TLC plates (Kieselgel 60 F254) were
purchased from Merck, Japan (Tokyo, Japan). All other reagents and
solvents were purchased from Nacalai Tesque (Kyoto, Japan). EI-MS
spectra were measured with an SX-102A spectrometer (JEOL, Tokyo,
Japan) or a GCQ gas chromatograph–mass spectrometer (Therm Fisher
Scientific, Yokohama, Japan). NMR spectra were recorded on an EX-
400 spectrometer (JEOL).
* Corresponding author (fax +81-88-656-7244; e-mail masuda@
ias.tokushima-u.ac.jp).
# University of Tokushima.
§ Nakazen, Inc.
10.1021/jf072893l CCC: $40.75
2008 American Chemical Society
Published on Web 12/15/2007

598 J. Agric. Food Chem., Vol. 56, No. 2, 2008
Masuda et al.
Tyrosinase Inhibition Assay. The tyrosinase inhibitory activity was
evaluated by using L-DOPA. The 96-well microplate method previously
reported by Masuda (24) was employed. Briefly, eight wells in total
were designated A (three wells), B (one well), C (three wells), and D
(one well), which contained the following reaction mixtures: A, 120
µL of a 1/15 M phosphate buffer (pH 6.8) and 40 µL of tyrosinase (46
units/mL) in the same buffer; B, 160 µL of the same buffer; C, 80 µL
of the same buffer, 40 µL of tyrosinase (46 units/mL) in the same buffer,
40 µL of an appropriate amount of the sample-buffer solution
containing 5% DMSO to dissolve the sample; D, 120 µL of the same
buffer and 40 µL of the same amount of the sample solution containing
5% DMSO. As a positive control experiment, kojic acid (final
concentration ) 0.03 mM) was used for a reference sample. The
contents of each well were mixed and then incubated at 23 °C for 10
min, before 2.5 mM L-DOPA in the same buffer was added. After
incubation at 23 °C for 2 min, the absorbance at 475 nm of each well
was measured. The percentage inhibition of the tyrosinase activity was
calculated according to the following equation: {[(A - B) - (C - D)]/
(A - B)} × 100. In the experiment for crude samples such as extract
and fraction, the addition orders of tyrosinase and L-DOPA was
inverted, because some phenolics in the extract reacted with tyrosinase
to interfere the oxidation of L-DOPA.
Extraction and Fractionation. The dry leaves of lemongrass (4
kg) were soaked in methanol (36 L) at room temperature for 2 weeks.
After filtration, the filtrate was evaporated under reduced pressure to
give a methanol extract (434 g). The extract (5.1 g) was partitioned
with hexane (50 mL) and methanol/water (9:1, 50 mL) to give a hexane-
soluble fraction and a methanol-soluble fraction. After removal of the
solvent, the methanol-soluble fraction was suspended in water (50 mL)
and sequentially extracted with ethyl acetate (50 mL) and n-butanol
(50 mL) to give an ethyl acetate-soluble fraction and a butanol-soluble
fraction, respectively. After evaporation, the yields of these fractions
were 1.5 g for the hexane extract, 1.3 g for the ethyl acetate extract,
and 0.7 g for the butanol extrtact. The remaining water layer was
evaporated to give the water extract (1.6 g). These extracts were used
for inhibitory assay.
From 200 g of the methanol extract was prepared 54 g of an active
ethyl acetate extract according to the same procedure as described
above. Ten grams of the ethyl acetate extract was loaded on a silica
gel column (600 mL) and eluted stepwise with 2.5% methanol in
chloroform (1.8 L), 5% methanol in chloroform (1.8 L), 10% methanol
in chloroform (1.8 L), 20% methanol in chloroform (1.2 L), and 30%
methanol in chloroform (1.2 L). The eluate was collected in every ca.
70 mL, and the obtained fractions (total of 109 fractions) were combined
to 21 fractions according to the similarity analysis for the constituents
of each fraction using silica gel TLC. The 21 fractions were subjected
to inhibitory assay after evaporation.
Figure 1. Chemical structures of geranic acid and related compounds.
Preparation of Ester Derivatives of Geranic Acid. To a solution
of geranic acid (201 mg, a mixture of trans and cis isomers) in
dichloromethane (7 mL) were added dimethylaminopyridine (73 mg),
dimethylaminopyridine hydrochloride (95 mg), ethanol (0.6 mL), and
dicyclohexylcarbodiimide (495 mg), sequentially. After 1 h of stirring
at 23 °C, acetic acid (0.2 mL) and methanol (0.4 mL) were added to
the mixture. After the addition of diethyl ether (40 mL), the produced
precipitate was filtered off. The filtrate was concentrated and subjected
to silica gel column chromatography eluted with chloroform/hexane
(1:6) to give geranic acid ethyl ester (3, 194 mg, 2:1 mixture of trans
and cis isomers): EI-MS, m/z 196 [M]+; 1H NMR (trans isomer)
(CDCl3) δ 1.28 (3H, t, J ) 7.5 Hz), 1.59 (3H, s), 1.67 (3H, s), 2.01
(7H, br s), 4.14 (2H, q, J ) 7.5 Hz), 5.06 (1H, m), 5.65 (1H, s).
A similar procedure using ethylene glycol instead of ethanol was
employed to synthesize geranic acid ethylene glycol ester (4, 76 mg,
2:1 mixture of trans and cis isomers) from geranic acid (105 mg): EI-
1
MS, m/z 212 [M]+; H NMR (trans isomer) (CDCl3) δ 1.60 (3H, s),
1.68 (3H, s), 2.15 (7H, br s), 3.84 (2H, m), 4.21 (2H, m), 5.07 (1H,
m), 5.71 (1H, s).
Preparation of an Amide Derivative of Geranic Acid. To the
hydroxysuccinimide ester of geranic acid (100 mg), which was prepared
from geranic acid, hydroxysuccinimide, and dicyclohexylcarbodiimide,
was added a chloroform solution (6 mL) of ethylamine hydrochrolide
(154 mg) and triethylamine (0.2 mL). After 20 min of stirring at 23
°C, the mixture was evaporated and subjected to silica gel column
chromatography eluted with ethyl acetate/hexane (1:4) to give geranic
acid ethyl amide (5, 32 mg, 2:1 mixture of trans and cis isomers): EI-
Isolation of Tyrosinase Inhibitory Substance. The most active
fraction 5 (1.1 g) of the above-mentioned silica gel chromatography was
purified again by silica gel column chromatography eluted with ethyl
acetate/hexane (1:4) to give 0.27 g of a fraction containing active substance.
To remove the lipidic impurity, 123 mg of the fraction was purified by
Sephadex LH-20 column chromatography eluted with isopropanol. Finally,
silica gel TLC purification (ethyl acetate/hexane ) 1:5) gave active
compounds 1 (14 mg, Rf ) 0.36) and 2 (5.5 mg, Rf ) 0.54).
1
MS, m/z 195 [M]+; H NMR (trans isomer) (CDCl3) δ 1.14 (3H, t, J
1
1 (trans-geranic acid): EI-MS, m/z 168 [M]+; H NMR (CDCl3) δ
) 7.2 Hz), 1.59 (3H, s), 1.68 (3H, s), 2.03–2.18 (4H, m), 2.12 (3H, s),
3.30 (2H, q, J ) 7.2 Hz), 5.06 (1H, m), 5.33 (1H, br), 5.53 (1H, s).
Preparation of Geranial and Farnesyl Aldehyde. To a solution
of geraniol (6, 306 mg) in hexane (15 mL) was added activated
manganese dioxide (4 g). After 4 h of stirring at room temperature,
the manganese dioxide was filtered off. The obtained filtrate was
evaporated and subjected to silica gel column chromatography eluted
with chloroform/hexane (1:1) to give geranial (7, 222 mg): EI-MS,
m/z 152 [M]+; 1H NMR (CDCl3) δ 1.62 (3H, s), 1.68 (3H, s), 2.17–2.28
(4H, m), 2.17 (3H, s), 5.07 (1H, m), 5.88 (1H, br t, J ) 7.5 Hz), 9.99
(1H, d, J ) 7.5 Hz).
According to a similar procedure, farnesyl aldehyde (14, 281 mg)
was synthesized from farnesol (400 mg): EI-MS, m/z 220 [M]+; 1H
NMR (CDCl3) δ 1.59 (6H, s), 1.67 (3H, s), 1.98 (2H, m), 2.04 (2H,
m), 2.15–2.28 (4H, m), 2.17 (3H, s), 5.08 (2H, m), 5.84 (1H, br d, J )
8.5 Hz), 9.98 (1H, d, J ) 8.5 Hz).
1.59 (3H, s, H-8 or 9), 1.66 (3H, s, H-8 or 9), 2.12 (3H, s, H-10), 2.13
(4H, m, H-4 and 5), 5.04 (1H, br s, H-6), 5.66 (1H, br s, H-2), 12.0
(1H, br, COOH); 13C NMR (CDCl3) δ 17.7 (C-9 or 10), 19.1 (C-9 or
10), 25.7 (C-8), 26.0 (C-5), 41.2 (C-4), 115.1 (C-2), 122.8 (C-6), 132.7
(C-7), 163.1 (C-3), 172.1 (C-1).
2 (cis-geranic acid): EI-MS m/z 168 [M]+; 1H NMR (CDCl3) δ 1.58
(3H, s, H-8 or 9), 1.66 (3H, s, H-8 or 9), 1.91 (3H, s, H-10), 2.14 (2H,
dt, J ) 8.0 and 7.0 Hz, H-5), 2.62 (2H, t, J ) 8.0 Hz, H-4), 5.12 (1H,
br t, J ) 7.0 Hz, H-6), 5.66 (1H, br s, H-2).
Preparation of trans- and cis-Geranic Acids (1 and 2). Com-
mercially available crude geranic acid (a mixture of mainly trans and
cis isomers) was separated by silica gel TLC (5 mg of sample loaded
on a 20 cm × 20 cm plate), which was developed twice with ethyl
acetate/hexane (1:5). From 44 mg of crude geranic acid were obtained
pure trans-geranic acid (1, 21 mg) and cis-geranic acid (2, 20 mg).

Geranic Acid in Lemongrass
J. Agric. Food Chem., Vol. 56, No. 2, 2008 599
Figure 2. Tyrosinase inhibitory activity and yield of the fractions by silica gel column chromatography of the ethyl acetate extract of the lemongrass. The
examined concentration of each fraction was 0.05 mg/mL, and the data were obtained in triplicate and averaged. Each SD value is expressed by an error
bar.
Table 1. Tyrosinase Inhibitory Activity (Percent Inhibition) of Geranic Acid and Related Compoundsa
concentration
compound
0.03 mM
0.1 mM
0.3 mM
1 mM
3 mM
IC50 (mM)
trans-geranic acid (1)
geranic acid ethyl ester (3)
geranic acid ethylene glycol ester (4)
geranic acid ethyl amide (5)
geraniol (6)
21.8 ( 1.8
43.6 ( 0.0
64.2 ( 1.0
82.4 ( 1.0
4.7 ( 2.7
-
0.14
#
#
#
#
1.62
1.70
#
#
#
-
-
1.8 ( 3.5
-
-
-
-8.3 ( 2.9
7.1 ( 2.2
-
-
-
-
-
-3.3 ( 2.3
-0.7 ( 1.2
41.4 ( 4.6
39.7 ( 5.6
4.5 ( 7.8
0.7 ( 3.0
-1.4 ( 3.2
60.3 ( 0.0
61.5 ( 0.0
8.3 ( 2.9
18.5 ( 1.3
-9.0 ( 1.1
-1.3 ( 2.2
7.4 ( 1.9
-
-
-
geranial (7)
-
8.0 ( 1.0
17.8 ( 2.6
3-methylcrotonic acid (8)
crotonic acid (9)
tiglic acid (10)
-
-
14.7 ( 1.1
-
-
-
-
-
-
-3.0 ( 3.4
-9.6 ( 1.9
-7.7 ( 0.0
4.3 ( 2.8
angelic acid (11)
-
-
-
methacrylic acid (12)
isovaleric acid (13)
farnesic acid (15)
-
-
-
#
#
0.11
-
-
-
23.0 ( 1.0
47.9 ( 1.0
70.9 ( 0.0
100 ( 0.0
a
-, not determined; #, could not be calculated.
Preparation of Farnesic Acid (All-trans Isomer). To a solution
of farnesyl aldehyde (14, 305 mg) in methanol (10 mL) were added
sodium cyanate (340 mg), acetic acid (119 µL), and activated
manganese dioxide (2.4 g). After 3 h of stirring at 23 °C, manganese
dioxide was filtered off. The filtrate was poured into ethyl acetate and
saturated NaCl aqueous solution and extracted three times with ethyl
acetate. The combined ethyl acetate layer was dried over anhydrous
sodium sulfate and evaporated. The residue was subjected to silica gel
chromatography eluted with chloroform/hexane (1:1) to give the methyl
ester of farnesic acid (a mixture of all-trans and 2-cis isomers, 184
mg). To the ester (105 mg) in methanol (5.5 mL) was added 1 N NaOH
aqueous solution. After 3 h of stirring at 60 °C, the mixture was poured
into 1 N HCl aqueous solution, extracted three times with chloroform,
and evaporated. The residue was purified by silica gel column
chromatography eluted with ethyl acetae/hexane (1:6) to give a cis and
trans mixture of farnesic acid (54 mg). Twenty milligrams of the mixture
was purified by silica gel TLC (ethyl acetate–/hexane ) 1:10) to give
(inhibitory percent for H, 28.7 ( 7.2; E, 62.5 ( 0; B, 55.2 (
3.5), whereas the water-soluble extract (W) promoted activity
(inhibitory percent for W, -41.4 ( 0). The ethyl acetate (E)-
and butanol (B)-soluble extracts showed potent activities
comparable to that of 0.03 mM kojic acid (inhibitory percent
) 70.1 ( 2.0). Thus, the most active ethyl acetate-soluble extract
was selected for the purification of the tyrosinase inhibitor of
the lemongrass. The ethyl acetate extract (54 g) was prepared
again from 200 g of the methanol extract according to the same
procedure, and 10 g of the extract was subjected to silica gel
column chromatography to separate into 21 fractions. The yield
and inhibitory activity of each fraction are summarized in Figure
1. Figure 1 shows that many fractions have a moderate
inhibitory activity at the concentration of 0.05 mg/mL. Among
the active fractions, the activity of fraction 5 was the strongest
and the yield was also the highest. The contribution of each
fraction to the activity of the plant should be estimated using
the value obtained by multiplication of each yield and activity;
therefore, fraction 5 should be selected as the fraction having
the greatest contribution and subjected to further investigation.
The constituents of fraction 5 were purified by subsequent silica
gel column chromatography, Sephadex LH-20 column chro-
matography, and silica gel TLC, to isolate the major compound
1 and the minor compound 2 as pure forms.
1
all-trans-farnesic acid (15, 14 mg): EI-MS, m/z 236 [M]+; H NMR
(CDCl3) δ 1.60 (6H, s), 1.70 (3H, s), 1.98 (2H, m), 2.05 (2H, m), 2.20
(4H, br), 2.20 (3H, s), 5.09 (2H, br), 5.70 (1H, s).
RESULTS AND DISCUSSION
Fractionation of the Extract of Lemongrass and Isolation
of Tyrosinase Inhibitors 1 and 2. The dry leaves of the
lemongrass (4 kg) were extracted with methanol, because the
alcoholic extract of the lemongrass showed a potent tyrosinase
inhibitory activity (5). The methanol extract (5.1 g) was next
separated into four extracts by a solvent partition method, and
the tyrosinase inhibitory activity of the extracts was measured.
Three organic solvent-soluble extracts (H, E, and B) showed
an inhibitory activity at the concentration of 0.15 mg/mL
Structure Identification and Tyrosinase Inhibitory Activi-
ties of 1 and 2. Compound 1 was isolated as a colorless oil. Its
molecular weight, which was obtained by EI-MS measurement,
and typical 1H and 13C NMR data, revealed that 1 was identical
to trans-geranic acid (25). Compound 2 was also isolated as a

600 J. Agric. Food Chem., Vol. 56, No. 2, 2008
Masuda et al.
colorless oil and showed the same molecular ion value as that
of 1. Analysis of 1H NMR data of 2 with comparison of that of
1 revealed that 2 was a cis isomer at the 2-olefin of 1 (25).
Therefore, 1 and 2 were identified as trans- and cis-geranic
acids, respectively (Figure 2).
The tyrosinase inhibitory activity of the trans- and cis-geranic
acids (1 and 2) was measured. trans-Geranic acid showed a clear
dose–response curve between 0.03 and 1 mM, and its IC50 value
was estimated to be 0.14 mM. Although the cis-geranic acid
also showed a dose–response curve, the activity of 2 was much
weaker than that of 1 (IC50 ) 2.3 mM). The activities of these
geranic acids were weaker than that of kojic acid (IC50 ) 0.017
mM), a potent tyrosinase inhibitor; however, the tyrosinase
inhibitory property of geranic acid was first reported in this
paper.
analogues. The conjugated double bond at the 2-position was
found to be essential because isovaleric acid (13), the single-
bond derivative of 8, showed almost no activity. Reduction or
rearrangement of the methyl substitution on the double bond
of 8 always reduced the activity, which was revealed from the
weak activities of crotonic acid (9), tiglic acid (10), angelic acid
(11), and methacrylic acid (12). This double-bond moiety would
cause a steric interaction with the tertiary structure of the active
site of tyrosinase when the carboxylic acid part is coordinated
to the active site. Therefore, the observed structural restriction
around the double bond would be required to increase the
inhibitory activity of geranic acid.
Geranic acid is a structurally simple tyrosinase inhibitor from
lemongrass; however, the very restricted structure of a long-
alkyl-substituted trans-conjugated carboxylic acid was estimated
as an essential structure for the potent tyrosinase inihibitory
activity.
Tyrosinase Inhibitory Activity of Geranic Acid Related
Compounds. Although geranic acid is a simple compound, the
stereochemical difference observed in the isolated trans and cis
isomers affects the inhibitory activity of geranic acid. A question
arose as to which part of the geranic acid structure was important
for the appearance of the tyrosinase inhibitory activity. There-
fore, the activity-required structure of geranic acid was analyzed
using several synthetic and commercially available analogs
(Figure 2). The percent inhibiton values of 12 geranic acid-
related compounds are summarized in Table 1. Geranial (7),
which was an aldehyde analogue of trans-geranic acid (1),
showed a lower activity than trans-geranic acid. Geraniol (6),
an alcohol analogue of trans-geranic acid, did not show any
activity under the examined conditions. The ester and amide
derivatives (3, 4, 5), which were synthesized from geranic acid,
showed no activity. These results indicated that the carboxylic
acid moiety of geranic acid was very important for its activity.
The tyrosinase inhibitory activity of various aromatic carboxylic
acids has been reported (26–30); however, these activity
strengths were lower than those of the structurally related
aldehyde derivatives (27, 28). In the case of the geranic acid
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structure, the carboxylic acid [trans-geranic acid (1), IC50
)
0.14 mM] was stronger than that obtained for the aldehyde
[geranial (7), IC50 ) 1.62 mM] (31). Most aldehyde inhibitors
are believed to show their activity by forming a stable Schiff
base with a primary amino group of the enzyme (32). On the
other hand, the carboxylic acids are suggested to inhibit by their
coordination to the copper of the binuclear site of the enzyme
(33). Geranic acid may have a structural part, which enhances
the coordination to the active site of the enzyme.
Next, to investigate the effect of the alkyl part adjacent to
the conjugated double bond (3-position) of geranic acid, farnesic
acid (15) was synthesized. Elongation of the C6 unit at the
3-position of geranic acid to the C11 unit, which appeared on
the fernesic acid, slightly increased the inhibitory activity (IC50
) 0.11 mM of 15 from 0.14 mM of 1). The reduction effect of
the C6 unit to the methyl group, which was examined using
3-methylcrotonic acid (8), reduced the activity (IC50 ) 1.70
mM). These results indicated that a longer alkyl chain adjacent
to the 3-position was effective, but not drastic. The appropriate
hydrophobic part of the enzyme inhibitor is well-known to
strengthen the activity by stabilizing its interaction with the
enzyme. For the tyrosinase inhibition, a similar effect has also
been observed (31, 34). In the case of geranic acid, the alkyl
part at the 3-position may contribute to this stabilization of the
enzyme–inhibitor interaction. Although the activity of 3-meth-
ylcrotonic acid (8) was weaker than that of trans-geranic acid,
it still has an attractive activity (IC50 ) 1.70 mM). Therefore,
the structural effect around the conjugated double bond at the
2-position was examined using the 3-methylcrotonic acid
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Received for review October 1, 2007. Revised manuscript received
November 13, 2007. Accepted November 18, 2007. This work was
financially supported by Kieikai Research Foundation (Tokyo, Japan).
JF072893L