Molecular design of antibrowning agents

Article abstract of DOI:10.1021/jf990926u

Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited the enzyme activity without being oxidized. This indicates that the carbon chain length is associated with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl (DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of side-chain length of gallates in relation to their antibrowning activity are studied.

Full text of DOI:10.1021/jf990926u

J. Agric. Food Chem. 2000, 48, 1393−1399
1393
Molecu la r Design of An tibr ow n in g Agen ts†
Isao Kubo,*,‡ Ikuyo Kinst-Hori,‡ Yumi Kubo,‡ Yoshiro Yamagiwa,§ Tadao Kamikawa,§ and
Hiroyuki Haraguchi|
Department of Environmental Science, Policy and Management, University of California,
Berkeley, California 94720-3112, Department of Chemistry, Faculty of Science and Technology,
Kinki University, Kowakae, Higashi-Osaka-shi, Osaka 577-8502, J apan, and Faculty of Engineering,
Fukuyama University, Gakuen-cho, Fukuyama-shi, Hiroshima 729-0292, J apan
Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were
investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by
mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as
substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited
the enzyme activity without being oxidized. This indicates that the carbon chain length is associated
with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein
pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed
potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl
(DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of
side-chain length of gallates in relation to their antibrowning activity are studied.
Keyw or d s: Tyrosinase inhibitors; antioxidants; antibrowning agents; molecular design; gallic acid;
dodecyl gallate
INTRODUCTION
usually not potent enough to be considered for a
practical use. A possible solution to cross this hurdle is
combining two or more inhibitors to increase the total
biological activity or their synthetic modification. The
emphasis in this paper was placed on an example for
the latter case. It should be noted however that safety
is a primary consideration for food additives since they
can be utilized in unregulated quantities. In our con-
tinuing search for mushroom tyrosinase inhibitors from
plants (Kubo, 1997), gallic acid was isolated from several
plants (Kubo and Matsumoto, 1985; Matsuo et al., 1997)
and identified as a potential PPO inhibitor. In addition,
gallic acid is a frequent constituent of hydrolyzable
tannins in many plants. On the other hand, the three
gallatesspropyl, octyl, and dodecylsare currently per-
mitted for use as antioxidant additives in food (Aruoma
et al., 1993); therefore, a series of its alkyl chain esters
was synthesized and their effects on mushroom tyrosi-
nase were investigated to determine if the gallates also
inhibit this enzyme. The aim of this research is to design
multifunctional antibrowning agents for foods.
In most foods, the browning process has two compo-
nents: enzymatic and nonenzymatic oxidation. This
unfavorable darkening from oxidation generally results
in a loss of nutritional and market values (Friedman,
1996). The enzymatic oxidation can be prevented by
tyrosinase (EC 1.14.18.1) inhibitors, and the nonenzy-
matic oxidation can be protected by antioxidants. Ty-
rosinase, also known as polyphenol oxidase (PPO)
(Zawistowski et al., 1991; Whitaker, 1995; Mayer and
Harel, 1998), is widely distributed in microorganisms,
animals, and plants. Plant PPO contributes negatively
to the color quality of plant-derived foods and beverages.
This unfavorable darkening from enzymatic oxidation
has been of some concern. It is responsible for not only
browning in plants but also melanization in animals.
For example, the unfavorable browning caused by
tyrosinase on the surface of seafood products has also
been of great concern (Ogawa et al., 1984). In addition,
tyrosinase inhibitors have become increasingly impor-
tant in medicinal (Mosher et al., 1983) and cosmetic
(Maeda and Fukuda, 1991) products in relation to
hyperpigmentation. Hence, tyrosinase inhibitors should
have broad applications.
MATERIALS AND METHODS
Gen er a l Meth od s. All the procedures used were the same
as previously described (Kubo and Kinst-Hori, 1998; Haraguchi
et al., 1997). UV-visible spectra were recorded by a Hitachi
100-80 spectrophotometer.
Ch em ica ls. Gallic acid and its methyl ester, L-DOPA,
pyrogallol, and DPPH were purchased from Aldrich Chemical
Co. (Milwaukee, WI) and were used as received. Xanthine
oxidase, L-tyrosine, propyl, ethyl, and dodecyl gallates, and
dimethyl sulfoxide (DMSO) were obtained from Sigma Chemi-
cal Co. (St. Louis, MO).
Although a large number of naturally occurring
tyrosinase inhibitors have already been described (Pifferi
et al., 1974; Mayer and Harel, 1979; Passi and Nazzaro-
Porro, 1981; Mayer, 1987), their individual activity is
* To whom correspondence should be addressed. Tel:
(510) 643-6303. Fax: (510) 643-0215. E-mail: ikubo@uclink4.
berkeley.edu.
† Dedicated to Prof. Dr. Takashi Kubota on the occasion of
his 90th birthday.
Syn th esis. To a solution of gallic acid (2 mM) and the
corresponding alcohol (2 mM) in THF (6 mL) cooled at 0 °C
was added a solution of dicyclohexylcarbodiimide (DCC) (4.2
mM) in THF (6 mL). After the solution had been allowed to
‡ University of California.
§ Kinki University.
| Fukuyama University.
10.1021/jf990926u CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/30/2000

1394 J. Agric. Food Chem., Vol. 48, No. 4, 2000
Kubo et al.
Ta ble 1. Tyr osin a se In h ibitor y Activity of Ga llic Acid
a n d Its Ester sa
stir for 20 h, the solvent was removed under reduced pressure.
The residue was extracted with ethyl acetate several times
and filtered. The filtrate was washed successively with dilute
aqueous citric acid solution, saturated aqueous sodium hydro-
gen carbonate solution, and water, dried (MgSO4), and evapo-
rated. The crude products were purified by chromatography
(SiO2; elution with chloroform-methanol, 98:2). Structures of
the synthesized esters were established by spectroscopic
methods (UV, IR, MS, and NMR). The best yield (87%) was
obtained with octyl gallate. It should be pointed out that
synthesis was achieved up to eicosanyl (C20) gallate but the
assay data were obtained unequivocally only up to dodecyl (C12)
gallate because of solubility problems of the esters having more
than 14 carbon atoms in the water-based test media. Among
the compounds tested unequivocally, dodecyl gallate was found
to be the most potent inhibitor. Therefore, the emphasis of
current study was centered on dodecyl gallate.
compounds tested
ID50 (mM)
gallic acid
4.5
C1
C3
C6
C8
C10
C12
0.35
0.31
0.21
0.33
0.28
0.49
a
With respect to L-DOPA.
of 2.28 mL of 2.51% linoleic acid in ethanol and 9 mL of 40
mM phosphate buffer (pH 7.0). The vial was placed in an oven
at 40 °C. At intervals during incubation, a 0.1 mL aliquot of
the mixture was diluted with 9.7 mL of 75% ethanol, which
was followed by adding 0.1 mM of 30% ammonium thiocyan-
ate. Precisely 3 min after the addition of 0.1 mL of 20 mM
ferrous chloride in 3.5% hydrochloric acid to the reaction
mixture, the absorbance at 500 nm was measured.
Ra d ica l Sca ven gin g Activity on DP P H. The reaction
mixture consisted of 1 mL of 100 mM acetate buffer (pH 5.5),
1 mL of ethanol, and 0.5 mL of ethanolic solution of DPPH.
After the mixture was allowed to stand at room temperature
for 20 min, the absorbance of the remaining DPPH was
determined colorimetrically at 517 nm. The scavenging activity
was measured as the decrease in absorbance of the DPPH
expressed as a percentage of the absorbance of a control DPPH
solution (Blois, 1958). Inhibitory activity was expressed as the
mean 50% inhibitory concentration of triplicate determina-
tions, obtained by interpolation of concentration-inhibition
curves.
Su p er oxid e An ion Assa y. The xanthine oxidase (XOD, EC
1.2.3.2) used for the bioassay was purchased from Sigma
Chemical Co. (St. Louis, MO). Superoxide anion was generated
enzymatically by xanthine oxidase system. The reaction
mixture consisted of 40 mM sodium carbonate buffer (pH 10.2)
containing 0.1 mM xanthine, 0.1 mM EDTA, 50 µg protein/
mL bovine serum albumin, 25 mM nitroblue tetrazolium
(NBT), and 3.3 × 10-3 units of xanthine oxidase in a final
volume of 3 mL. After incubation at 25 °C for 20 min, the
reaction was terminated by the addition of 0.1 mL of 6 mM
CuCl2. Since superoxide anion reduces yellow NBT to blue
formazan, the generation of superoxide anion can be detected
by measuring the absorbance of formazan newly produced at
560 nm (Toda et al., 1991).
Hexyl ga lla te was obtained in 56% yield as a colorless
solid: 1H NMR (270 MHz) δ 0.88 (t, J ) 6.5 Hz, 3H), 1.22-
1.46 (m, 6H), 1.65-1.76 (m, 2H), 4.18-4.28 (br, 2H), 6.4-5.8
(br, 3H, exchangeable with D2O), 7.2 (s, 2H); IR (KBr) 3389,
2932, 1687, 1614, 1448 cm-1
.
Octyl ga lla te was obtained in 87% yield as a colorless
solid: 1H NMR (270 MHz) δ 0.88 (t, J ) 6.5 Hz, 3H), 1.20-
1.48 (m, 13H), 1.64-1.78 (m, 2H), 4.24 (t, J ) 6.5 Hz, 2H),
7.15 (s, 2H); IR (KBr) 3325, 2926, 1686, 1614, 1468 cm-1
.
Decyl ga lla te was obtained in 65% yield as a colorless
solid: 1H NMR (270 MHz) δ 0.88 (t, J ) 6.5 Hz, 3H), 1.22-
1.47 (m, 15H), 1.66-1.78 (m, 2H), 5.24 (t, J ) 6.5 Hz, 2H),
7.16 (s, 2H); IR (KBr) 3391, 2924, 1686, 1514, 1448 cm-1
.
Tyr osin ase Assay. The mushroom tyrosinase (EC 1.14.18.1)
used for the bioassay was purchased from Sigma Chemical Co.
(St. Louis, MO). Although mushroom tyrosinase differs some-
what from other sources (van Gelder et al., 1997), this fungal
source was used for the entire experiment because it is readily
available. It should be noted that the commercial tyrosinase
was described to contain numerous proteins besides tyrosinase
(Kumar and Flurkey, 1991) but was used without further
purification. The preliminarily assay of the samples was tested
at 167 µg/mL. The samples were first dissolved in DMSO and
used for the experiment at 30-fold dilution. The enzyme
activity was monitored by dopachrome formation at 475 nm
up to the appropriate time (usually not exceeding 10 min,
unless otherwise specified). Tyrosinase catalyzes a reaction
between two substrates, a phenolic compound and oxygen, but
the assay was carried out in air-saturated solutions.
The tyrosinase assay was performed as previously described
(Masamoto et al., 1980) with some modification. First, 1 mL
of a 2.5 mM L-DOPA or L-tyrosine solution was mixed with
1.8 mL of 0.1 M phosphate buffer (pH 6.8) and incubated at
25 °C for 10 min. Then, 0.1 mL of the sample solution and 0.1
mL of the aqueous solution of the mushroom tyrosinase (138
units, added last) were added to the mixture to immediately
measure the initial rate of linear increase in optical density
at 475 nm.
The preincubation mixture consisted of 1.8 mL of 0.1 M
phosphate buffer (pH 6.8), 0.6 mL of water, 0.1 mL of the
samples solution (equivalent amount of ID50), and 0.1 mL of
the aqueous solution of the mushroom tyrosinase (138 units).
The mixture was preincubated at 25 °C for 5 min. Then, 0.4
mL of 6.3 mM L-DOPA solution was added, and the reaction
was monitored at 475 nm for 2 min.
For the measurement of the visible (350-550 nm) spectra
of the oxidation products produced by mushroom tyrosinase,
the mixture consisting of 1.8 mL of 0.1 M phosphate buffer
(pH 6.8), 1.0 mL of water, or 2.5 mM L-DOPA solution was
preincubated at 25 °C for 10 min. Subsequently, 0.1 mL (7.5
mM) of the sample solution and 0.1 mL of the aqueous solution
of the enzyme (138 units, added last) were added, and the
mixture was incubated at 25 °C for 3min. Then, the spectra
were recorded.
RESULTS AND DISCUSSION
Tyr osin a se In h ibitor y Activity. The inhibitory
activity of gallic acid (1) and its esters differing in their
alkyl chain lengths tested with respect to the oxidation
of L-DOPA catalyzed by mushroom tyrosinase is listed
in Table 1. As opposed to gallic acid, ID50 values of all
the esters are almost comparable. The bioassay with
gallic acid showed a concentration-dependent inhibitory
effect on this oxidation, and the ID50 was determined
as 767 µg/mL (4.5 mM). This ID50 is about 7-fold less
potent than that of benzoic acid, a well-documented
tyrosinase inhibitor (Pifferi et al., 1974; Wilcox et al.,
1985; Conrad et al., 1994). In addition, gallic acid itself
served as a substrate and was oxidized by the enzyme.
This oxidation was characterized by a new peak with
the maximum at 383 nm, presumably corresponding to
o-quinone absorption (Passi and Nazzaro-Porro, 1981),
but at a much slower rate without a cofactor. There was
a progressive increase in the observed rate of this slow
oxidation as soon as catalytic amounts of L-DOPA
became available as a cofactor, and a yellowish color
was immediately detected optically. As long as a cofactor
was present, gallic acid was oxidized faster than L-
Au toxid a tion Assa y. Oxidation of linoleic acid was mea-
sured as previously described (Osawa and Namiki, 1981). The
test samples dissolved in 120 µL were added to a reaction
mixture in a screw cap vial. Each reaction mixture consisted

Antibrowning Agents
J. Agric. Food Chem., Vol. 48, No. 4, 2000 1395
F igu r e 3. Mixing effect of gallic acid (4.5 mM): (O) without
gallic acid; (b) without gallic acid and with mixing; (4) with
gallic acid; (2) with gallic acid and with mixing.
F igu r e 1. Inhibitory effect on the oxidation of L-DOPA
catalyzed by mushroom tyrosinase: (O) without and (b) with
gallic acid (4.5 mM).
F igu r e 2. Structure of gallic acid (1) and its possible structure
of its primary oxidation product (2) catalyzed by tyrosinase.
F igu r e 4. Lineweaver-Burk plots of mushroom tyrosinase
and L-DOPA without (4) and with dodecyl gallate [(2) 0.18
mM and (9) 0.3 mM]. 1/V: 1/∆475 nm/min.
DOPA since the peak at 383 nm reached its plateau
faster than that of 475 nm. This may be the reason gallic
acid was reported as a tyrosinase inhibitor and also a
substrate (Passi and Nazzaro-Porro, 1981; Andrawis
and Kahn, 1990; Matsuo et al., 1997). Because of its
enzymatically oxidizable nature, the mode of inhibition
of gallic acid could not be analyzed. It should be noted
that the assay was carried out in air-saturated aqueous
solutions. Therefore, after several minutes, dopachrome
formation reached the plateau as all the available
oxygen in the cuvette was consumed. As shown in
Figure 1, the inhibitory curve lowered compared to the
control curve and the difference of “a” indirectly dem-
onstrates the amount of oxygen in the cuvette used for
the oxidation of gallic acid. The oxidation products are
a complex mixture of polar compounds, and the unstable
nature of the intermediates makes their characteriza-
tion difficult. Despite our efforts, an attempt to char-
acterize them failed. However, the resulting o-quinone
may condense with one another through a Michael-type
addition, yielding a quinol-quinone product (2) (Sayre
and Nadkarni, 1994) (Figure 2), though the possibility
that the o-quinone may form adducts with different
nucleophilic groups in the enzyme and inactivates it
cannot be entirely ruled out. The former case seems to
be more likely since the remaining L-DOPA in the
cuvette was oxidized when oxygen was supplied by
mixing as illustrated in Figure 3. This result indicates
that the enzyme was not inactivated by a Kcat-type
inhibition (inactivation of the enzyme by products of
the reaction), as long as the current experiment is
concerned. It appears that gallic acid was isolated as a
tyrosinase inhibitor by bioassay-guided fractionation
using mushroom tyrosinase similar to the previous
report (Matsuo et al., 1997), but this phenolic acid can
also be termed as an alternate substrate if enough
oxygen is supplied.
Similarly, methyl (C1) gallate was characterized as
an inhibitor but also served as an alternate substrate.
That is, this gallate was oxidized by the enzyme at a
slow rate and yielded the colored oxidation products.
This slow oxidation rate was also significantly increased
when catalytic amounts of L-DOPA became available as
a cofactor. Similar to those observed with gallic acid,
the difference of a in the inhibition curve of methyl
gallate indirectly demonstrates the amount of oxygen
in the cuvette used for the oxidation of methyl gallate.
However, in contrast to gallic acid, the oxidation prod-
ucts of methyl gallate did not show noticeable absorption
around 380 nm. It is obvious therefore that the oxidation
products, a complex mixture of polar compounds, were
different from those of gallic acid, presumably the
resulting o-quinone immediately undergoes a series of
nonenzymatic reactions. In addition, similar results
were also obtained with up to nonanyl (C9) gallates,
though yields of the yellowish oxidation products were
decreased with increasing the carbon number.
Interestingly, dodecyl (C12) gallate inhibited the oxi-
dation of L-DOPA catalyzed by mushroom tyrosinase,
but without yielding the colored products. The ID50 was
determined as 147 µg/mL (0.49 mM), which is nearly
11-fold more potent compared to that of gallic acid. The
inhibition kinetics of dodecyl gallate was analyzed by a
Lineweaver-Burk plot as shown in Figure 4. The result
indicates that dodecyl gallate exhibited as a mixed-type
inhibitor with respect to the oxidation of L-DOPA
catalyzed by mushroom tyrosinase. In addition, prein-
cubation of the enzyme in the presence of 0.49 mM of

1396 J. Agric. Food Chem., Vol. 48, No. 4, 2000
Kubo et al.
F igu r e 6. Mixing effect of dodecyl gallate (0.49 mM): (O)
without dodecyl gallate; (b) without dodecyl gallate and with
mixing; (4) with dodecyl gallate; (2) with dodecyl gallate and
with mixing.
F igu r e 5. Inhibitory effect on the oxidation of L-DOPA
catalyzed by mushroom tyrosinase: (O) without and (b) with
dodecyl gallate (0.49 mM).
dodecyl gallate and in the absence of L-DOPA did not
decrease the enzyme activity significantly. Similar to
those observed with the short alkyl chain esters afore-
mentioned, the inhibition curve of dodecyl gallate
indicates that the oxygen in the cuvette was used in part
for the oxidation of dodecyl gallate as illustrated in
Figure 5. This observation may indicate that dodecyl
gallate was oxidized to the corresponding o-dihydroxy
derivative by the enzyme without further oxidation to
the yellowish o-quinone. The resulting o-quinone usually
undergoes secondary reactions, either between quinones
or with other substances in the immediate vicinity, and
a variety of colored compounds are formed consequently
(J anovitz-Klapp et al., 1990). In the current experiment,
the pigmented products did not appear up to 2 h,
indicating dodecyl gallate was not oxidized to the
corresponding o-quinone. The alternative possibility
that dodecyl gallate is oxidized to the o-quinone, which
oxidizes other products with lower redox potentials, and
is immediately reduced back to the colorless o-dihydroxy
derivative cannot be totally ruled out because tyrosinase
usually oxidizes o-diphenols to o-quinones. However,
this possibility is unlikely. Or, the resulting o-quinone
inactivates the enzyme without producing the pig-
mented products, but this possibility is even more
unlikely. Most probably, dodecyl gallate is not oxidized
under the current experimental conditions and inhibits
the oxidation of L-DOPA, presumably by being coordi-
nated to copper as a substrate analogue and positioned
over the binuclear site (Winkler et al., 1981; Conrad et
al., 1994). This likely suggests that dodecyl gallate is
an inhibitor rather than an inactivator of the enzyme
(Kahn and Andrawis, 1985). The above assumption can
be supported by the observation that the remaining
L-DOPA in the cuvette was oxidized when oxygen
became available by mixing as shown in Figure 6. The
inhibition activity exerted by dodecyl gallate could be,
at least in part, on the basis of the assumption that the
corresponding oxygen a can be held as the peroxide in
the oxy-form of tyrosinase [Eoxy] to which dodecyl gallate
binds as an inhibitor [I] and the resulting peroxo-
inhibitor complex [EoxyI] is inactive. On the basis of the
above observation, it may be reasonable to conclude that
the gallates with increasing the carbon number of the
alkyl group (>C10) may become harder to be embraced
by the protein pocket (Tanford, 1980; Wilcox et al., 1985)
and as a result decrease the rate of oxidation by the
enzyme. Hence, the gallates with the longer alkyl group
(>C10) become inhibitors but not substrates. This indi-
cates that the gallates with the longer alkyl group
(>C10) can be expected as more appropriate inhibitors.
However, tetradecanyl gallate (C14) and hexadecanyl
gallate (C16) tested are hardly soluble in the water-based
test media. This caused variation with O.D. readings
that were essential in determining the ID50. Therefore,
their ID50 could not be established unequivocally. The
precise explanation for the role of the alkyl chain
lengthswhich must be related to their tyrosinase
inhibitory activitysstill remains obscure. However, it
is apparent that the oxidation rate of the gallates by
the enzyme decreases with increasing the alkyl chain
length. In other words, the gallates with increasing
hydrophobicity of the molecules become more resistant
to being oxidized by the enzyme. On the basis of this
observation, it may not be illogical to assume that more
hydrophobic interaction with the enzyme increases
disruption of the tertiary structure of the enzyme and
increases the resistance to being oxidized.
Tyrosinase contains a strongly coupled binuclear
copper active site and functions both as a monopheno-
lase and as an o-diphenolase (Lerch, 1987; Sa´nchez-
Ferrer et al., 1995). The discussion so far described is,
however, on the basis of the experiment using L-DOPA
as a substrate. Therefore, the activity aforementioned
is o-diphenolase inhibitory activity of mushroom tyro-
sinase. In the absence of a cofactor, the hydroxylation
of monophenols such as L-tyrosine to L-DOPA is char-
acterized by an initial lag period. Some reductants
added exogenously can either reduce or abolish the lag
period (Prota, 1992). o-Dihydroxyphenols have been
described as the most efficient cofactors for the hydrox-
ylation of monophenols (Pomeranz and Warner, 1967).
Interestingly, the lag period for the hydroxylation of
L-tyrosine was eliminated in the presence of pyrogallol,
but not gallic acid. This indicates that gallic acid does
not serve as a cofactor. It is worthwhile to add that the
gallate esters, regardless of their carbon chain length,
slightly shorten the lag time, indicating they serve as a
weak cofactor.
As far as the ID50 values are compared, the alkyl
chain length of the gallates seems to be not related to
their mushroom tyrosinase inhibitory activity. However,
gallic acid and its short alkyl (<C10) chain esters were
oxidized by tyrosinase as substrates yielding the yellow
oxidation products, but the long alkyl (>C10) chain
esters inhibited the enzyme without producing the
pigmented products, indicating that the carbon chain
length is related to their tyrosinase inhibitory activity
in different ways. Although gallic acid and its esters

Antibrowning Agents
J. Agric. Food Chem., Vol. 48, No. 4, 2000 1397
Ta ble 2. An tioxid a tive Activity of Ga llic Acid a n d Its
Ester s
IC50 (µM)
gallates tested
DPPH radical
O2•- generation
gallic acid
C1
C2
C3
C6
C8
C10
C12
2.86
3.73
4.41
4.23
4.82
6.39
4.82
6.39
5.94
9.51
10.30
11.49
25.15
>100
>100
>100
radical scavenging activity, which can be measured as
the decolorizing activity following the trapping of the
unpaired electron of DPPH, was examined. Gallic acid
and its esters assayed showed almost equally potent
radical scavenging activity. The complete scavenging
activity was obtained in the range of 10-30 µM, and
their 50% scavenging concentrations are listed in Table
2.
It is well-established that lipid peroxidation is one of
the reactions set into motion as a consequence of the
formation of free radicals in cells and tissues. The one-
electron reduction products of O2, superoxide anion
(O2•-), hydrogen peroxide (H2O2), and hydroxy radical
(HO•) actively participate in the initiation of lipid
peroxidation. Several oxidative enzymes, such as xan-
F igu r e 7. Antioxidative activity of (2) gallic acid, (b) dodecyl
gallate, (4) R-tocopherol, and (O) control. Each compound was
added at a final concentration of 30 µg/mL.
were reported as substrates of mushroom tyrosinase
(Passi and Nazzaro-Porro, 1981), the long alkyl (>C10)
chain esters can be used as tyrosinase inhibitors since
they do not yield the pigmented products. A similar
result was recently reported that the long alkyl (>C12)
chain gallates prevent cell damage induced by hydroxyl
radicals or hydrogen peroxides (Masaki et al., 1997). A
more hydrophobic alkyl (>C12) chain gallates may
provide the unique ability to be positioned in the cell
membrane, similar to the phytyl chain in tocopherols
and tocotrienols (Papas, 1999). It appears that the
current study using mushroom tyrosinase is a simple
but effective strategy for the initial screening to search
for antibrowning agents. Needless to add, other experi-
mental factors such as the reaction time and amount of
oxygen are needed to be considered from a practical
point of view. In connection with this, the previous
report that mushroom tyrosinase differs somewhat from
other sources (van Gelder et al., 1997) also needs to be
borne in mind.
An tioxid a n t Activity. Unsaturated fatty acids, es-
pecially linoleic acid, are the target of lipid peroxidation.
The effect of gallic acid and dodecyl gallate on autoxi-
dation of linoleic acid was measured by the ferric
thiocyanate method as previously described (Osawa and
Namiki, 1981). The result is shown in Figure 7. In a
control reaction, the production of lipid peroxide in-
creased almost linearly during 8 days of incubation.
R-Tocopherol, also known as vitamin E, at 30 µg/mL
inhibited the linoleic acid peroxidation almost 50%. Both
gallic acid and dodecyl (C12) gallate were found to be
more effective in preventing this oxidation than R-to-
copherol. About 50% inhibition was observed at 10 µM
with dodecyl gallate. Dodecyl gallate was found to show
more potent activity than gallic acid, indicating the
dodecyl group is not essential but related to the activity.
All the esters tested, regardless of their carbon chain
length, exhibited antioxidant activity as expected. Mem-
brane lipids are abundant in unsaturated fatty acids.
These unsaturated molecules are most susceptible to
oxidative processes, particularly linoleic acid. Lipid
peroxidation is a typical free-radical oxidation and
proceeds via a cyclic chain reaction (Witting, 1980). The
•-
thine oxidase, produce the O2 radical as a normal
product of the one-electron reduction of oxygen, result-
ing in tissue injury (Mayumi et al., 1993). The scaveng-
ing activity of gallic acid and its esters against enzymic-
generated superoxide anion by xanthine oxidase system
is shown in Table 2. Among them, gallic acid exhibited
the most potent scavenging activity with IC50 of 5.9 µM,
and the long alkyl (>C10) chain gallates did not show
any activity up to 100 µM, indicating that the alkyl
chain length is not associated with this activity.
SUMMARY
In addition to their autoxidation inhibitory activity,
all the gallate esters tested, regardless of their carbon
chain length, showed potent scavenging activity on the
DPPH radical, indicating that the alkyl chain length is
not related to this activity. Therefore, all these esters
can be considered as antioxidants. The result is in
agreement with the current use of gallatesspropyl,
octyl, and dodecylsas antioxidant additives in foods and
previous report (Gunckel et al., 1998). If the gallates
need to be used only as antioxidants, the selection
depends on their physical properties, such as boiling
point and solubility. It should be kept in mind, however,
that methyl and propyl gallates were reported to exert
prooxidant effects toward DNA and carbohydrates by
interacting with iron (Aruoma et al., 1993). In addition,
the short alkyl (<C10) chain esters were oxidized them-
selves by tyrosinase and produced the pigmented oxida-
tion products, so it appears that the long alkyl (>C10)
chain gallates can be considered as more applicable
tyrosinase inhibitors with antioxidant activity. The
complete scavenging activity of gallic acid was observed
in the range of 10-30 µM that is much less than the
amounts needed to use as a tyrosinase inhibitor. This
potent scavenging activity should be of considerable
advantage to control enzymatic pigmentation, since
radicals are involved in melanin synthesis. For example,
superoxide anion was reported to enhance the oxidation

1398 J. Agric. Food Chem., Vol. 48, No. 4, 2000
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