Pharmaceutical Biology
2007, Vol. 45, No. 6, pp. 519–524
Antityrosinase Activity of Some Plant Extracts and Formulations
Containing Ellagic Acid
Ö. Özer1, B. Mutlu1, and B. Kıvçak2
Department of Pharmaceutical Technology; 2Department of Pharmacognosy, Faculty of Pharmacy, Ege University,
Bornova, Izmir, Turkey
1
Abstract
Ellagic acid (EA) is a naturally occurring polyphenol
found in a variety of plants in its free form or in the form
of ellagitannin glycosides. In this study, the ellagic acid
content of the methanol extracts of Juglans regia L.
(Juglandaceae) leaves, Castanea sativa Mill. (Fagaceae)
stem bark, and Eucalyptus camaldulensis Dehnh.
(Myrtaceae) leaves was determined to develop melanogenesis inhibitors. An improved NaNO2 assay was used
for determination of EA. The tyrosinase inhibitory
activity of the extracts and synthetic EA was tested in
vitro by monitoring the appearance of dopachrome, an
intermediate in the melanogenesis process. The results
were compared keeping the same total concentration of
inhibitor. The efficacy of EA (1%) was compared with
arbutin (1%) and hydroquinone monomethyl ether
(1%) as reference substances, and it was found to be a
more efficient suppressor of pigmentation. The effect
of formulation variables on the tyrosinase inhibitory
activity was also evaluated. Based on dopachrome
tests performed in the formulations, it could be concluded that the combination with plant extracts had a
synergistic effect, and gel formulation could be suggested as an effective carrier for treating uneven skin
pigmentation.
Keywords: Castanea sativa stem bark, ellagic acid, Eucalyptus camaldulensis leaves, Juglans regia leaves, topical
formulations, tyrosinase.
Introduction
Hyperpigmentation is the most common facial pigmentary disorder. It has been observed that a local increase
in melanin synthesis or uneven distribution of melanin
can cause local hyperpigmentation or spots. Whitening
agents such as hydroquinone, arbutin, kojic acid, or
azelaic acid are widely used in cosmetic products as
active substances (Prota, 1996; Mora & Baraldi, 2000;
Nakayama et al., 2000; Petit & Piérard, 2003).
Plant extracts that have a good inhibitory effect on
melanin formation may be a good choice for the cosmetic
purposes of whitening facial skin and protection against
skin darkening. In addition, they have relatively fewer
side effects (Kim & Lee, 1998). In cosmetic preparations,
many plant extracts such as Morus alba L. (Moraceae) or
Glycyrrhiza glabra Linneva. (Leguminosae) have been
used as whitening agents (Lee et al., 1997; Bernard &
Berthon, 2000; Baurin et al., 2002).
Tyrosinase is the enzyme involved in melanogenesis
and catalyzes the oxidation process of tyrosine to
dihydroxy-phenylalanine (DOPA) and from DOPA to
DOPA quinone. Tyrosinase is known to be a metaloenzyme, containing copper at an active site, catalyzing
these reactions through a change in the oxidative site
of copper atoms (Lee & Kim, 1995; Prota, 1996; Mora
& Baraldi, 2000).
Ellagic acid (EA) is a naturally occurring polyphenol
found in a variety of plants in its free form or in the form
of ellagitannin glycosides. It exists in dicotyledonous
woody plants, in the genera of Castanea (Fagaceae),
Eucalyptus (Myrtaceae), and Quercus (Fagaceae). It is
also found in a variety of fruits and vegetables normally
consumed by humans such as grapes, cherry, and walnut
(Zee-Cheng & Cheng, 1986; Peng et al., 1991; Bianco
et al., 1998; Lei et al., 2001; Amakura et al., 2002).
Recently, EA was shown to inhibit skin pigmentation
resulting from UV irradiation. Results of in vitro
Accepted: January 18, 2007.
Address correspondence to: Özgen Özer, Assoc. Prof. Dr., Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege
University, 35100 Bornova, İzmir, Turkey. Fax: þ 90232 3885258; E-mail: ozgen.ozer@ege.edu.tr
DOI: 10.1080/13880200701446746
# 2007 Informa Healthcare
520
Ö. Özer et al.
experiments have indicated that EA suppresses melanogenesis by inhibiting tyrosinase activity. This inhibition
is caused by chelation of the copper atoms on the tyrosinase molecules. It was approved in 1996 for use as an
active ingredient in formulations for the prevention of
spots and freckles after excessive exposure to sunlight
(Shimogaki & Yanagawa, 1996; Shimogaki et al., 2000;
Tanaka, 2001).
The aim of this study was to determine the EA content
and tyrosinase inhibitory effect of Folium Juglandis
(Juglans regia leaves), Cortex Castanea (Castanea sativa
stem bark), and Folium Eucalypti (Eucalyptus camaldulensis leaves) methanol extracts to develop melanogenesis
inhibitors. An improved NaNO2 assay was used for
determination of EA (Wilson & Hagerman, 1990).
Tyrosinase inhibitory effects of the plant extracts were
compared with the synthetic EA by measuring the
melanin intermediates (dopachrome) spectrophotometrically (Ferioli et al., 2001). After this study, the tyrosinase
inhibitory activity of different formulations containing
synthetic EA and plant extract was also studied to determine the effect of formulation variables.
Materials and Methods
Plant materials and preparation of plant extracts
Castanea sativa Mill. (Fagaceae) stem bark and Juglans
regia L. (Juglandaceae) leaves were collected from Sultanhisar (Malgeçemir village) in Aydın in September
2002. Eucalyptus camaldulensis Dehnh. (Myrtaceae)
leaves were also collected from the Ege University campus
in September 2002 and identified by B. Kivcak. Voucher
specimens (no. 1275, no. 1277, no. 1276 in order) were
deposited at the herbarium of the Faculty of Pharmacy,
Ege University, in Izmir.
Plant materials were air-dried and then powdered.
Each powdered plant (100 g) was extracted with 80%
methanol solution, and after filtration the filtrates
were evaporated to dryness under vacuum. These
extracts were used for further studies including quantitative determination of ellagic acid and inhibition of
tyrosinase.
Drugs and chemicals
EA was obtained from Shangai Pudong New Area Li
Cheng Group (China). L-Tyrosine and mushroom tyrosinase (EC 1.4.18.1., activity 127 U=mg of solid) were
purchased from Fluka (Sigma-Aldrich, Italy). Methanol
was obtained from J. T. Baker (Holland). The oil used
was liquid paraffin (Birpa, Turkey). The lipophilic surfactants were Abil EM 90 (Goldschmid, France) and
Span 80, a sorbitan oleat (ICI, France), and the hydrophilic surfactants were Tween 80, a polyoxyethylene
sorbitan oleat, and Synperonic PE= F 127, an ethoxylated
propylene oxide copolymer (ICI). Carbopol 954, a synthetic polymer, and Carsoquat CT 429 were obtained
from Johnson & Johnson (Turkey). Triethanolamine
(TEA) was from Sigma (St. Louis, MO, USA). All other
chemicals used for analysis were analytical reagent grade.
Quantitative determination of EA from the plant extracts
The quantity of free EA existing in natural sources was
determined with an improved NaNO2 assay method that
was used by Wilson and Hagerman (1990). This assay
was first described by Bate-Smith (1972) and was selective for ellagic acid only; gallic acid, gallotannins, ellagitannins, condensed tannins, and flavonoids do not
interfere.
Different concentrations of EA in dimethyl sulfoxide
were added to dimethyl sulfoxide to give a final volume
of 9.2 mL in test tubes. Concentrated HCl (0.4 mL) was
then added and samples were brought to 30C. The samples were mixed immediately after 0.4 mL 1% (w=v)
NaNO2 in H2O was added. The absorbance at 513 nm
was recorded, and equation of linear calibration curve
was obtained.
For determination of EA from plant extracts, 10 mg of
extract was dissolved in 10 mL of DMSO. This stock solution (1 mL) was assayed for EA using the described
NaNO2 method.
Determination of tyrosinase inhibitory effect of synthetic
active materials and plant extracts
Tyrosinase inhibitory activity is generally determined
with a spectrophotometer. The procedure described by
Vanni et al. (1990) was followed in our study. Tyrosinase
inhibitory activity of EA was determined and compared
with the tyrosinase inhibitory activity of arbutin (Nihei &
Kubo, 2003) and hydroquinone monomethyl ether
(HQMME) (Patrick et al., 1999) as reference substances.
Tyrosinase inhibition effect of Cortex Castanea extract,
Folium Eucalypti extract, and Folium Juglandis extract
was also determined.
For evaluation of tyrosinase inhibitory effect, a reaction mixture consisted of 0.9 mL 50% methanol solution
of inhibitor and 2 mL L-tyrosine solution (0.244 mM) in
aqueous phosphate buffer (pH ¼ 6.8; I ¼ 0.01 M) was
prepared; for the control sample, an equivalent volume
of 50% methanol solution was used instead of the inhibitor solution. Oxidation of L-tyrosine was initiated by
introducing 0.1 mL of aqueous mushroom tyrosinase solution (0.1 mg=mL). Test mixture and control mixture
were incubated for 10 min at 37C. Dopachrome appearance was monitored spectrophotometrically at 475 nm.
We determined the effect of test compound on tyrosinase
inhibition by IC50, the concentration at which the
compound inhibits half the original tyrosinase activity.
Antityrosinase activity of plants containing ellagic acid
521
The percent inhibition of tyrosinase activity was calculated as follows:
% inhibition :
ðAcontrol
Asample
Þ
475
475
Acontrol
475
100
and Asample
were the absorbance values in the
Acontrol
475
475
presence and absence of inhibitor. Tyrosinase inhibitory
activities of plant extracts were evaluated with the same
method using the same mixture without plant extract
as a control.
Preparation of skin-whitening formulations
For investigation of the tyrosinase inhibitory effect of
formulations, an oil=water (O=W) type emulsion (F1),
a W=O=W type multiple emulsion (F2), a gel (F3), a
lotion (F4), and a vanishing cream (F5) containing synthetic ellagic acid, plant extracts, and a combination of
these were prepared.
Determination of tyrosinase inhibitory effect
of formulations
Blank and spiked cream was weighed accurately into a
centrifuge tube (1 g). Ten millilitres of 50% methanol
was added and stirred in an ultrasonic bath for 30 min.
The remaining suspension was centrifuged at 4000 rpm
for 30 min. Aliquots of the resultant solution were
further diluted 1:10 with 50% methanol and tested by
the dopachrome method for determination of tyrosinase
inhibitory effect.
Statistical analysis
Tests for significant differences between mean values
were made by analysis of variance (ANOVA). Reference
to significant difference in the following text denotes that
the test was carried out at a level of p < 0.05.
Figure 1. Tyrosinase inhibitory activity of ellagic acid (&),
arbutin (.), and HQMME (~) at different (0.1 mM, 0.3 mM,
0.5 mM) concentrations (n ¼ 6, SD).
sources can be done by acid hydrolysis of the crude tannin extracts. (Zee-Cheng & Cheng, 1986; Peng et al.,
1991; Bianco et al., 1998; Amakura et al., 2002). However, the products of acidic hydrolysis process are
thought to be a disadvantage for cosmetic products.
For this reason, in this study methanol extracts of the
plant materials were prepared and the quantity of free
ellagic acid was determined spectrophotometrically
(Table 1). Folium Juglandis extract was found to have
the highest concentration of EA. EA content of Folium
Juglandis, Cortex Castanea, and Folium Eucalypti
extracts were found to be 16.25%, 2.75%, and 0.28%,
respectively.
The tyrosinase inhibitory effects of arbutin and
HQMME, which are well-known whitening agents,
were determined to prove the accuracy of our method.
Suppression of tyrosinase could be demonstrated when
dose-dependent inhibition was demonstrated using these
hydroquinone derivatives as an effective control. The
Results and Discussion
It is known that ellagic acid naturally occurs in Eucalyptus camaldulensis, Castanea sativa, and Juglans regia
at high concentrations in its free form or in the form of
ellagitannin. Isolation of ellagic acid from natural
Table 1. The quantity of EA in the plant extracts
(n ¼ 6 SD).
Plant sample
Folium Eucalypti extract
Cortex Castanea extract
Folium Juglandis extract
EA%
0.28 0.021
2.75 0.335
16.25 0.512
Figure 2. Tyrosinase inhibitory activity of Folium Juglandis
extract (&), Cortex Castanea extract (.) and Folium Eucalypti
extract (~) at different (100 mg=mL, 250 mg=mL, 500 mg=mL)
concentrations (n ¼ 6, SD).
522
Ö. Özer et al.
Table 2. Concentration (IC50) required for selected tyrosinase
inhibitors to reduce mushroom tyrosinase activity by 50%.
IC50
(mg=mL)
Plant extracts
Folium Juglandis extract
Cortex Castanea extract
Folium Eucalypti extract
505
844
2258
inhibitory activity was estimated starting from test
compound solutions at 0.1 to 0.5 mM.
As expected, a concentration-dependent inhibitory
effect was observed for each compound tested as shown
in Figure 1. The results indicate that the more the concentration of compounds was increased, the more the
tyrosinase inhibitory capacity increased.
The inhibitory effect of arbutin and HQMME
was compared with EA, and except for HQMME at
0.5 mM, significant difference was observed for all concentrations (p < 0.05). The results demonstrated that
all of the synthetic active materials showed an inhibitory
activity level higher than 50% at the concentration of
0.5 mM, and among these synthetic materials, EA has
shown the highest tyrosinase inhibitory effect.
IC50 values of the compounds were also compared,
and EA was found to be a more potent tyrosinase inhibitor than arbutin and HQMME. IC50 values were
calculated as 0.225, 0.311, and 0.267 mM for EA, arbutin, and HQMME, respectively.
According to the results of previous studies conducted
using the brownish guinea pig, EA was found to be a
more efficient skin whitener and suppressor of pigmentation than arbutin or kojic acid at the same dose level
(1%). Furthermore, the efficacy of EA was found almost
the same to that of HQMME, a well-known depigmentation agent. These results are in good accordance with our
results (Patrick, 1999; Shimogaki et al., 2000).
The isolation and structural determination of the
ellagic acid from plant extracts will provide us new
information leading to the development of new skinwhitening products. Current attempts to find new
Table 3. Compositions of the skin-lightening formulations.
F1
(g)
Formulation
20
Mineral oil
(Paraffinum
liquidum)
Span 80
Tween 80
Triethanolamine
Abil EM 90
Synperonic PE=F 127
MgSO4 7H2O
Propylene glycol
Carbopol 954
Methyl paraben
Cetyl alcohol
Stearic acid
Lanoline
Glycerine
Carsoquat CT 429
Potassium hydroxide
Distilled water
F2
(g)
F3
(g)
F4
(g)
24
F5
(g)
10
1.75
3.25
0.3
4
0.8
0.7
49
2
0.15
0.75
0.5
3
1
2
2
72.75
70.5
53.85
86.35
1
24
10
1.2
63.8
skin-whitening agents focus on the systematic evaluation
of plant extracts. We investigated the inhibitory effect of
Folium Eucalypti (Eucalyptus camaldulensis), Cortex
Castanea (Castanea sativa), and Folium Juglandis
(Juglans regia) extracts on tyrosinase activity for this
purpose. These three plant extracts, which demonstrated
a significant ability to inhibit mushroom tyrosinase, were
described for the first time to possess this biological property. Dopachrome test was performed on these plant
extracts at three different concentrations. Folium Juglandis
extract exhibited the highest inhibition of tyrosinase at
the concentration of 500 mg=mL (Fig. 2). The inhibitory
effect of Folium Juglandis was found to be 1.2- and 1.8fold higher than that of Folium Eucalypti and Cortex
Castanea extracts, respectively, for all concentrations.
The presence of the free form of EA at a high concentration seems to verify the high tyrosinase inhibition of
Folium Juglandis extract. When we compared the inhibitory effect of Folium Eucalypti and Cortex Castanea with
Table 4. Combinations that were incorporated into the formulations.
Combination
C1
C2
C3
C4
C5
C6
C7
C8
Ellagic
acid (1%)
Folium Eucalypti
extract (1%)
Folium Juglandis
extract (1%)
Cortex Castanea
extract (1%)
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Antityrosinase activity of plants containing ellagic acid
523
Table 5. Tyrosinase inhibitory effect of formulations (n ¼ 6 SD).
Combination
C1
C2
C3
C4
C5
C6
C7
C8
F1
(%)
F2
(%)
F3
(%)
F4
(%)
F5
(%)
43.262 0.512
6.117 0.323
13.475 0.597
19.167 0.466
45.783 0.812
51.950 0.908
58.777 0.840
62.450 0.105
38.021 0.615
6.337 0.422
15.017 0.718
17.649 0.469
39.843 0.875
47.830 0.831
54.295 0.717
58.631 0.925
47.553 0.845
6.329 0.557
19.590 0.498
22.779 0.714
51.539 0.918
59.11 0.771
62.450 0.955
66.529 0.720
39.545 0.986
4.594 0.678
17.235 0.671
21.396 0.791
41.257 0.919
44.257 0.991
51.181 0.928
56.405 0.935
37.743 0.714
5.115 0.529
18.959 0.564
21.718 0.559
40.740 0.861
51.181 0.964
52.910 0.521
58.082 0.743
Folium Juglandis, a significant difference was observed
for all concentrations (p < 0.05), only the difference with
Cortex Castanea at 250 mg=mL was found insignificant
(p > 0.05).
When IC50 values of the compounds were compared,
Folium Juglandis extract was found to be more effective,
reaching the IC50 at a much lower concentration than
Folium Eucalypti and Cortex Castanea extracts (Table 2).
(Castanea sativa), and Folium Juglandis (Juglans regia)
extracts on tyrosinase activity were described for the first
time for this biological property. The highest amount of
EA and the strongest inhibitory effect on tyrosinase were
observed with Juglans regia extract, followed by Eucalyptus camaldulensis and Castanea sativa extracts. These
plant extracts could be suggested as new sources of
skin-whitening agents.
Tyrosinase inhibitory effect of formulations
Composition of the formulations and combinations that
were introduced into the formulations are given in
Tables 3 and 4. Ellagic acid (1%), plant extract (1%),
and combinations of these were introduced into each
formulation.
Evaluation of the formulation effects on tyrosinase
inhibition demonstrated that the gel formulation containing synthetic EA and three plant extracts together
at 1% concentration has the highest inhibitor activity
(approximately 66.6%). Methanol extract of blank formulations was used as control sample, and no inhibitory
effect caused by the formulation was observed. Tyrosinase inhibitory effects of formulations are given in Table 5.
Comparison of the inhibitory activity of the formulations demonstrated that the rank order was approximately the same, F3 > F1 > F2 > F5 > F4, for all
combinations. The results of the formulations F2, F5,
and F4 were found similar. The inhibitor activity was
found higher for gel formulation than for that obtained
with the other formulations in all cases. In addition,
the combinations C8 and C1 showed the highest inhibitor activity in all formulations, respectively. Comparison
of F3-C8 formulation with other formulations containing C8 combination indicated that there were statistically
significant differences between gel formulation and the
others (p < 0.05).
According to the results, it was suggested that gel formulation containing ellagic acid and three plant extracts
together will be effective in treating uneven skin pigmentation. In conclusion, the inhibitory effect of Folium
Eucalypti (Eucalyptus camaldulensis), Cortex Castanea
Acknowledgment
We gratefully acknowledge the Ege University Research
Foundation for its financial support.
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