A novel ring-expanded product with enhanced tyrosinase inhibitory activity from classical Fe-catalyzed oxidation of rosmarinic acid, a potent antioxidative Lamiaceae polyphenol

Article abstract of DOI:10.1016/j.bmcl.2010.10.040

The iron-ion catalyzed oxidation of the ethanol solution of rosmarinic acid, a potent antioxidant polyphenol of Lamiaceae (Labiatae) plants, afforded a highly tyrosinase-inhibitory active product. The structure of the active product in the oxidation product mixture was determined using extensive NMR spectroscopy to have a novel oxygen-containing seven-membered ring system. The formation mechanism of the unique ring structure from the catechol part of the rosmarinic acid was proposed.

Full text of DOI:10.1016/j.bmcl.2010.10.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7393–7396
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel ring-expanded product with enhanced tyrosinase inhibitory
activity from classical Fe-catalyzed oxidation of rosmarinic acid,
a potent antioxidative Lamiaceae polyphenol
Aya Fujimoto a, Yoshimi Shingai a, Mitsuhiro Nakamura a, Tomomi Maekawa b,
Yoshiaki Sone b, Toshiya Masuda a,
⇑
a Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan
b Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The iron-ion catalyzed oxidation of the ethanol solution of rosmarinic acid, a potent antioxidant polyphe-
nol of Lamiaceae (Labiatae) plants, afforded a highly tyrosinase-inhibitory active product. The structure of
the active product in the oxidation product mixture was determined using extensive NMR spectroscopy
to have a novel oxygen-containing seven-membered ring system. The formation mechanism of the
unique ring structure from the catechol part of the rosmarinic acid was proposed.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 August 2010
Revised 17 September 2010
Accepted 8 October 2010
Available online 14 October 2010
Keywords:
Rosmarinic acid
Polyphenol
Antioxidant
Tyrosinase inhibitory activity
Fe-catalyzed oxidation
Structure determination
Natural polyphenol has currently attracted much attention as
an active constituent in functional foods and food supplements,
which promises to improve human health. The polyphenol shows
a very potent antioxidant activity, and this activity is closely linked
to various beneficial actions, including anti-aging, prevention of
cancer, cardiovascular disease, etc.1 The antioxidant efficiency of
the polyphenolic antioxidant depends on its oxidizable property.
The potent antioxidant is oxidized much faster than other biomol-
ecules, thus providing the potent antioxidant activity.2 This easily
oxidizable property of the polyphenol may lead to new oxidation
products in foods and in the human body.3–8 However, the func-
tionality, regardless of being beneficial or non-beneficial, of the
oxidation products has not yet been intensively examined.
Recently, we examined the chemical structures and cytotoxic
property of the oxidation products from sesamol, a sesame antiox-
idant.9 We have now screened tyrosinase inhibitory activity of the
oxidation products from various polyphenols. Tyrosinase is widely
distributed in plants, microorganisms, and animals. In food, tyros-
inase accelerates the browning of fresh food, which results in a loss
of its market value. In humans, tyrosinase is responsible for the
melanization of skin. Therefore, tyrosinase inhibitors have at-
tracted strong interest in food and cosmetic sciences.
Rosmarinic acid (1) is a potent antioxidative polyphenol widely
distributed in Lamiaceae (Labiatae) herbs.10 The Lamiaceae herbs
such as rosemary, sage, and melissa contain a large amount of ros-
marinic acid and they are used as a food additive and herb tea for
multifunctional purposes. Many beneficial activities of rosmarinic
acid, which include antiinflammation,11 antimutagenicity,12
photoprotection of keratinocytes,13 reduction of atopic dermati-
tis,14 prevention of Alzheimer’s disease,15 apoptosis induction of
colorectal cancer cell,16 etc., have been recently reported in
addition to its potent antioxidant activity.17 Rosmarinic acid
1
1"
O
O
COOH
OH
O
O
COOH
O 2'
2
3"
3
A
4' O
5' O
6'
OH
O
B
3"'
HO
HO
4"'
OH
OH
2
1
⇑
Corresponding author. Tel.: +81 88 656 7244; fax: +81 88 656 7244.
E-mail address: masuda@ias.tokushima-u.ac.jp (T. Masuda).
Figure 1. The structure of rosmarinic acid (1) and its oxidation product (2).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.040

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A. Fujimoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7393–7396
Table 1
1H, 13C and 2D NMR data for 2a
Posn.
dH (m, Hz)
Correlated H in long range COSYb
Correlated H in NOESY
dC
Correlated H in HMBC
1
2
3a
173.0
71.2
28.5
H2, H3ab
H3ab
5.31 (dd, 8.5, 4.5)
2.88 (dd, 14.0, 8.5)
3.13 (dd, 14.0, 4.5)
H3ab
H3ab, H30, H60
H2, H30, H60
H2, H60
H2, H60, H3b
H2, H60, H3a
H2, H60
3b
10
127.0
180.6
118.6
160.2
155.2
156.7
64.0
H3ab, H30, H60
H3ab, H30, H60
20
30
7.03 (br s)
H60
H2, 40-OCH2CH3
40
H30, 400-OCH2CH3
H30, H60
H3ab
50
60
8.22 (s)
4.38 (q, 7.0)
1.36 (t, 7.0)
H3ab,H30
40-OCH2CH3
40-OCH2CH3
H2, H3ab
H30, 40-OCH2CH3
40-OCH2CH3
40-OCH2CH3
40-OCH2CH3
14.2
100
168.2
114.1
148.0
127.6
115.2
146.8
149.8
116.7
123.2
H2, H200, H300
H300
200
6.26 (d, 15.5)
7.56 (d, 15.5)
H300
300
H200, H2000, H5000, H6000
H200, H2000, H6000
H200, H300, H2000, H5000
H5000, H6000
1000
2000
3000
4000
5000
6000
7.03 (br s)
H300, H5000, H6000
H200, H300
H2000, H5000
H2000, H5000, H6000
H2000, H6000
6.77 (d, 8.0)
6.95 (dd, 8.0, 2.0)
H300, H2000, H6000
H300, H2000, H5000
H6000
H200, H300, H5000
H300, H5000
a
Recorded in CD3OD at 400 or 500 MHz for 1H and 125 MHz for 13C.
Used delay time was 200 ms.
b
should be one of the promising food-functional polyphenols
(Fig. 1).
to those of the rosmarinic acid. In the 1H NMR of 2 (Table 1), only
two singlet signals were observed as assignable to the ring A
protons, both of which were correlated in long range COSY spec-
trum of 2. The chemical shift of the benzylic proton signals of ring
A were slightly changed from that of rosmarinic acid. These results
indicated that a new ring system was produced on the original ring
A of rosmarinic acid by this Fe-catalyzed oxidation. The fine
structure of the ring A was mainly deduced by the C–H long range
coupling connectivities observed in the HMBC spectra of 2 (Fig. 2).
Two aromatic proton signals (d 8.22 and 7.03), and two benzylic
proton signals (d 2.88 and 3.13) were correlated with the carbon
at 180.6 ppm, which indicated that the 2-position of ring A should
be a carbonyl group. The aromatic proton at 7.03 ppm was also cor-
related with the carbons at 160.2 and 155.2 ppm, one of which
(160.2 ppm) was adjacent to the ethoxyl group which was revealed
by an HMBC correlation with the methylene protons (d 4.38). The
other aromatic proton at 8.22 ppm was also correlated with the
1-position carbon (127.0 ppm), a benzylic carbon (28.5 ppm), and
the carbon at 155.2 ppm in the HMBC spectrum (Fig. 2) and with
the carbon at 156.7 ppm in the HMQC spectrum. The HR-MS re-
sults revealed that ring A contained C8H7O4 in the molecular for-
mula (C20H18O10). These results strongly indicated that the ring A
has a unique oxygen-contained seven-membered ring structure
as shown in Figure 1. A very upfield shifted carbonyl carbon at
155.2 ppm of 2 should be assignable to the 5-position of ring A
by the HMBC correlations and comparison of the typically upfield
shifted signal of the 2-carbonyl carbon observed in 3-oxygenated
coumarins (ex. 158.5 ppm), which had the similar conjugated sys-
tem to that of 2.18
To obtain the oxidation products of rosmarinic acid, we
employed the Fe-catalyzed oxidation as the possible oxidation
reaction in nature including foods. To an ethanol solution of ros-
marinic acid was added a catalytic amount of FeCl3, and stored un-
der an oxygen atmosphere until almost all the rosmarinic acid was
consumed. The resulting oxidation mixture was found to show a
remarkable tyrosinase inhibitory activity compared to that of the
oxidation products from other antioxidative polyphenols and re-
lated phenolics (caffeic acid, catechin, carnosic acid, carnosol,
chlorogenic acid, ellagic acid, epicatechin, eugenol, gallic acid, gen-
tisic acid, gingerol, hydroxytyrosol, nordihydroguaiaretic acid,
piceatannol, protocatechuic acid, propyl gallate, quercetin, resvera-
trol, sinapic acid, syringic acid, secoisolariciresinol, taxifolin, and
a-tocopherol). Although the oxidation product from rosmarinic
acid contained various constituents, our assay-guided purification
of the active mixture resulted in the isolation of one active com-
pound having a novel oxidized structure. In this communication,
we report the unique chemical structure and its potent tyrosinase
inhibitory activity.
The oxidation of rosmarinic acid was carried out as follows. To
100 mg of rosmarinic acid in ethanol (10 mL) was added 0.05 M
FeCl3 (0.05 mL) in
a
straight screw-capped vial (i.d. 35 ꢀ h
78 mm). Ten such vials were incubated at 40 °C for 5 days under
an oxygen atmosphere. The solutions were combined and then
treated with Chelex 100™ (20 g) to remove the Fe ions and evapo-
rated. The residue was fractionated by column chromatography
using an ODS gel (Cosmosil-140C18-OPN) eluted with 20–70%
methanol in H2O containing 0.1% TFA. The 30% methanol-eluted
fraction was purified by HPLC (Column: Cosmosil 5C18-AR-II,
20 ꢀ 250 mm, Solvent: methanol–1% AcOH aq = 1:1) to afford an
active compound (2, 32 mg, tR 12 min). The HR-ESI-MS of 2 (m/z
417.0818 [MꢁH]ꢁ) suggested C20H18O10 as the molecular formula
of 2. Although the 1H NMR of 2 was very similar to that of rosmari-
nic acid (1), the signal set due to an ethoxy group was additionally
observed, indicating that 2 was a reacted compound with the sol-
vent from the original rosmarinic acid. The reacted position of the
ethanol was indicated to be on the aromatic ring A because the caf-
feoyl (including aromatic ring B) and 2-oxypropionic acid moieties
were intact from their proton and carbon NMR signals very similar
O
COOH
O
R
CH
H
H2C
2'
4'
5'
OCH2CH3
6'
H
O
O
2
: R=Caffeyl
Figure 2. Proton-carbon long range correlations (arrows) around the A ring of 2 in
HMBC spectrum.

A. Fujimoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7393–7396
7395
O
O
COOH
O
O
COOH
O
O
COOH H+
O OH
OOH
OH
OH
OH
O
OH
O
HO
HO
HO
OH
OH
Fe2+
OH
1
3
4
Fe3+
O2
O2
O
O
COOH
O
O
COOH
O
O
O
COOH
O
H
O
H
OH
OH
H+
OEt
O
H+
OEt
O
O
O
HO
HO
HO
EtOH
OH
OH
O
OH
O
5
2Fe3+
2Fe2+
O2
H2O2
O
O
COOH
O
O
COOH
O
O
COOH
O
OEt
OEt
OH
O
OEt
H+
O
O
HO
O
HO
OH
H+
HO
OH
OH
OH
2
HOOH
6
Figure 3. A proposed mechanism for formation of 2 from rosmarinic acid (1) in the presence of Fe ion.
The Fe-catalized oxidation afforded the unique ring-expanded
compound from rosmarinic acid, however, its isolation yield was
very low. Therefore we explored the possibilities that this reaction
generally occurred in the catechol structure. Ethyl 3-(3,4-dihy-
droxyphenyl)-2-hydroxypropionate, which was a simplified ester
derivative of rosmarinic acid by removing oxidatively sensitive caf-
feyl moiety of rosmarinic acid, was oxidized under the same condi-
tions, yielding the similar seven-membered ring compound19 in
over 30% yield without any adjustment of the reaction conditions.
Further investigation to record higher yield of the oxidation is now
in progress. This oxidation also took place in 4-methyl catechol and
the produced the seven-membered compound20 showed two
carbonyl absorptions at 1651 cmꢁ1 (conjugated carbonyl part)
and 1747 cmꢁ1 (ester part) in IR spectrum21 and two carbonyl sig-
nals at 181.8 ppm and similar upfield-shifted 154.8 ppm in 13C
NMR to that of 2, supporting the seven-membered structure of 2
and its generality of the reaction of catechol compounds. It should
be noted that this type of ring-expanding oxidation of catechol is
proposed in the reaction of human homogentisate dioxygenase
by Titus and co-workers.22 Our found reaction maybe a biomimetic
oxidation and should support Titus’ proposed enzymatic reaction
mechanism. In Figure 3, one of the possible formation mechanisms
of the unique structure of 2 from rosmarinic acid (1) was illus-
trated. At first, the catechol structure of the ring A of rosmarinic
acid was oxidized by ferric ion to produce the phenolic radical 3.
The produced ferrous ion reduces oxygen to produce a superoxide
anion. In the second step, the rosmarinic acid radical 3 is reacted
with a protonated superoxide radical to afford a hydroperoxide 4.
This hydroperoxide is unstable and then converted to diketoepox-
ide 5 by a mechanism similar to the epoxidation reaction of an en-
Table 2
Tyrosinase inhibitory activity of rosmarinic acid (1), oxidation mixture from rosmarinic acid, and compound 2a
Compound
Concentration
0.30 mM
IC50 (mM)
0.01 mM
0.03 mM
0.10 mM
1.00 mM
3.00 mM
10.0 mM
b
Compound 2
10.6 2.6
—
—
35.3 0.9
—
—
52.5 1.0
—
32.6 2.9
—
—
—
—
—
—
0.08
1.40
0.25
0.012
Rosmarinic acid (1)
Oxidation mixture from 1c
Kojic acidd
40.7 8.2
63.8 5.9
—
69.8 13.0
—
—
93.6 2.5
—
—
58.7 3.1
—
47.8 3.4
74.1 1.7
a
b
c
Each value is expressed as mean SD (n = 3).
— Not examined.
Molar concentration was calculated as rosmarinic acid (MW 360).
Positive control sample.
d

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A. Fujimoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7393–7396
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2009, 50, 3905.
one by alkaline hydroperoxide. Several migration reactions with
the addition of ethanol produce paraquinone 6. The quinone 6 is
then reacted with a hydroperoxide via the Baeyer–Villiger reaction
to afford compound 2.
The tyrosinase inhibitory activity of 2 was evaluated based on a
previously reported procedure using mushroom tyrosinase.23 Com-
pound 2 showed the inhibitory activity between 0.01 and 0.1 mM
to be concentration-dependent and its IC50 was calculated to be
0.08 mM. The IC50 values of rosmarinic acid and its oxidation mix-
ture under the same conditions were obtained to be 1.4 and
0.25 mM, respectively. Therefore, compound 2 has a very strong
inhibitory activity and should be chemical evidence of the very
strong activity of the oxidation product of rosmarinic acid (Table
2).
10. Petersen, M.; Simmonds, M. S. J. Phytochemistry 2003, 62, 121.
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Acknowledgments
1
19. H NMR (acetone-d6, 400 MHz): d 8.10 (1H, s, H60), 6.92 (1H, s, H30), 4.39 (2H, q,
J = 7.2 Hz, 40-OCH2), 4.37 (1H, overlapped with the signal of 40-OCH2, H2), 4.13
(2H, q, J = 7.2 Hz, 1-OCH2), 2.9 (1H, overlapped with the signal of H2O, H3b),
2.62 (1H, dd, J = 14.0 and 8.0 Hz, H3a), 1.36 (3H, t, J = 7.2 Hz, 40-OCH2CH3), 1.12
(3H, t, J = 7.2 Hz, 1-OCH2CH3); 13C NMR (CDCl3, 100 MHz): d 179.3 (C20), 173.7
(C1), 159.7 (C40), 154.0 (C60), 152.9 (C50), 126.4 (C10), 118.4 (C30), 68.7 (C2),
63.0(40-OCH2), 61.9 (1-OCH2), 30.6 (C3), 14.1 (1- or 40-OCH2CH3), 14.0 (1- or 40-
OCH2CH3); HR-ESI-MS m/z 293.0630 [M+Na]+, calcd for C12H14O7Na, 293.0637.
Proton and carbon numbering system was adopted temporarily form that of 2.
This project was financially supported by a Grant from Uehara
Memorial Foundation (Tokyo). We express thanks to Mr. Hirose
and Mr. Hayashi of Waters Japan for their help to perform
HR-ESI-MS (Xevo-QTOF MS spectrometer).
References and notes
1
20. H NMR (CD3OD, 400 MHz): d 8.06 (s, H60), 6.87 (s, H30), 4.38 (2H, q, J = 7.2 Hz,
40-OCH2), 1.87 (3H, s, H1), 1.36 (40-OCH2CH3); 13C NMR (CD3OD, 100 MHz): d
181.8 (C20), 161.1 (C40), 155.3 (C60), 154.8 (C50), 128.3 (C10), 117.9 (C30), 64.0
(40-OCH2), 14.3 (40-OCH2CH3), 10.8 (C1); HR-ESI-MS m/z 183.0650 [M+H]+,
calcd for C9H11O4, 183.0657. Proton and carbon numbering system was
adopted temporarily form that of 2.
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