Antioxidation mechanism of rosmarinic acid, identification of an unstable quinone derivative by the addition of odourless thiol

Article abstract of DOI:10.1016/j.foodchem.2011.11.062

The antioxidation mechanism of rosmarinic acid was investigated. An AIBN-induced oxidation reaction of linoleate in the presence of rosmarinic acid produced one main product. The DPPH reaction of rosmarinic acid also gave two products, one of which corresponded to the product of the lipid system. Direct isolation of the products failed because of their instability. To the DPPH reaction, the addition of 1-dodecanethiol afforded three isolable adducts. Their structures revealed that the reaction produced quinone derivatives on either the caffeoyl or 2-oxyphenylpropanoyl moieties. However, the lipid oxidation afforded one quinone, which could convert to a thiol adduct. The HPLC of the reaction clarified that the antioxidation product of rosmarinic acid was the unstable quinone on the 2-oxyphenylpropanoyl moiety. This quinone formation was the first stage of the antioxidation of rosmarinic acid in the lipid system. The odourless 1-dodecanethiol is a useful reagent to identify the unstable quinone from polyphenol oxid ation.

Full text of DOI:10.1016/j.foodchem.2011.11.062

Food Chemistry 132 (2012) 901–906
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Antioxidation mechanism of rosmarinic acid, identification of an unstable
quinone derivative by the addition of odourless thiol
⇑
Aya Fujimoto, Toshiya Masuda
Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 August 2011
Received in revised form 19 October 2011
Accepted 14 November 2011
Available online 20 November 2011
The antioxidation mechanism of rosmarinic acid was investigated. An AIBN-induced oxidation reaction of
linoleate in the presence of rosmarinic acid produced one main product. The DPPH reaction of rosmarinic
acid also gave two products, one of which corresponded to the product of the lipid system. Direct
isolation of the products failed because of their instability. To the DPPH reaction, the addition of 1-
dodecanethiol afforded three isolable adducts. Their structures revealed that the reaction produced qui-
none derivatives on either the caffeoyl or 2-oxyphenylpropanoyl moieties. However, the lipid oxidation
afforded one quinone, which could convert to a thiol adduct. The HPLC of the reaction clarified that the
antioxidation product of rosmarinic acid was the unstable quinone on the 2-oxyphenylpropanoyl moiety.
This quinone formation was the first stage of the antioxidation of rosmarinic acid in the lipid system. The
odourless 1-dodecanethiol is a useful reagent to identify the unstable quinone from polyphenol
oxidation.
Keywords:
Antioxidation
Reaction mechanism
Rosmarinic acid
Quinone
Odourless thiol
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
1997; Masuda et al., 2008; Moon & Terao, 1998). Although various
antioxidative caffeic acid derivatives, including chlorogenic acid
Rosmarinic acid (1) is a potent antioxidative polyphenol widely
distributed in Lamiaceae herbs (Petersen & Simmonds, 2003). The
Lamiaceae herbs, such as rosemary, sage, and melissa, contain a
large amount of rosmarinic acid and are used as a food additive
and herbal tea for multifunctional purposes in worldwide food
cultures. Many beneficial functionalities of rosmarinic acid, which
include anti-inflammation (Huang et al., 2009), anti-mutagenicity
(Furtado, de Almeida, Furtado, Cunha, & Tavares, 2008), the
photoprotection of keratinosytes (Psotova, Svbodova, Kolarova, &
Walterova, 2006), reduction of atopic dermatitis (Lee, Jung, Kog,
Kim, & Park, 2008), prevention of Alzheimer’s disease (Hamaguchi,
Ono, Murase, & Yamada, 2009), apoptosis induction of colorectal
cancer cells (Xavier, Lima, Fernandes-Ferreira, & Pereira-Wilson,
2009), etc., have been recently reported in addition to its potent
antioxidant activity (Nakatani, 1996). Rosmarinic acid is now recog-
nised to be one of the promising food-functional polyphenols. With
respect to its chemical structure, rosmarinic acid has two catechol
moieties. Catechol is an important sub-structure for the potent anti-
oxidant activity of phenolic antioxidants. In rosmarinic acid, one
catechol structure exists in the caffeoyl moiety and the other is in
the 2-oxyphenylpropanoyl moiety. It is well known that caffeic acid
has been recognised as a very strong antioxidant and its antioxidant
mechanism has already been intensively investigated (Chen & Ho,
and caffetaric acid, are contained in various edible plants and
plant-derived foods (Shahidi & Naczk, 2004), their antioxidation
mechanism has not yet been fully understood, because of their
structural complexity compared to that of caffeic acid.
For the general antioxidation mechanism of phenolic antioxi-
dants, the process is thought to be divided into two stages as
shown by the following schemes (Frankel, 2005):
(1) radical trapping stage
R ꢀ OO radical þ AH ! R ꢀ OOH þ A radical
(2) radical termination stage
A radical ! non-radical materials
where R is the substance for oxidation, the R-OO radical is the
peroxyl radical of R, AH is the antioxidant, and the A radical is the
antioxidant radical. Although the first stage is a reversible process,
the second stage is irreversible and must produce a non-radical
product. The structure of the product would afford important infor-
mation about the antioxidation mechanism of the phenolic antioxi-
dants (Masuda et al., 2010). The antioxidation process of rosmarinic
acid against biomolecule oxidation, such as unsaturated lipid oxida-
tion, is very attractive for clarifying not only its antioxidation mech-
anism of caffeic antioxidants, but also its relation to its various
⇑
Corresponding author. Fax: +81 88 656 7244.
E-mail address: masuda@ias.tokushima-u.ac.jp (T. Masuda).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.11.062

902
A. Fujimoto, T. Masuda / Food Chemistry 132 (2012) 901–906
bioactivities. In this study, the antioxidation product from rosmari-
nic acid in a lipid oxidation system was investigated. To clarify the
antioxidation product from rosmarinic acid, a useful and generally
applicable method to identify the produced unstable quinone deriv-
ative was newly developed using an odourless thiol reagent.
Tesque); solvent, 1% acetic acid in H2O/CH3OH = 9:1, flow rate,
9.5 ml minꢀ1; detection, 280 nm, to give fraction 1 (14 mg) at the
retention time (9 min) and fraction 2 (82 mg) at the retention time
(11 min). Fraction 1 was purified again by preparative HPLC under
the following new conditions, column, Cosmosil 5C18-AR-II
(20 ꢁ 250 mm); solvent, 1% acetic acid in H2O/CH3OH = 85:15, flow
rate, 9.5 ml minꢀ1; detection, 280 nm, to give compound 4 (8 mg).
Fraction 2 (82 mg) was purified by a recycle preparative HPLC
under the following new conditions, column, Cosmosil 5C18-AR-
II (20 ꢁ 250 mm); solvent, 1% acetic acid in H2O/CH3OH = 70:30,
flow rate, 9.5 ml minꢀ1; detection, 280 nm, recycle 5 (5 mg) and
6 (27 mg).
2. Materials and methods
2.1. Chemicals and instruments
Rosmarinic acid (1) was purchased from Aldrich Japan (Tokyo,
Japan). 2,20-Azobis(isobutyronitrile) (AIBN) and 1-dodecanethiol
were obtained from Tokyo Kasei (Tokyo, Japan). Ethyl linoleate
was purchased from Kanto Chemicals (Tokyo, Japan) and used after
purification by silica gel (silica 60, Merck, Darmstat, Germany)
chromatography developed with 2.5% ethyl acetate in hexane. All
solvents and other reagents were obtained from Nacalai Tesque
(Kyoto, Japan). The NMR spectra were measured using an
ECS-400 spectrometer (JEOL, Tokyo, Japan) with the manufacturer-
supplied pulse sequences [1H, 13C, correlated spectroscopy (HH-
COSY) and nuclear Overhauser effect spectroscopy (NOESY)]. The
mass spectra were measured with XEVO Qtof spectrometers
(Waters Japan, Tokyo, Japan) in the negative ESI mode. A LC-
20ATvp low pressure gradient system (Shimadzu, Kyoto, Japan)
equipped with a photodiode array detector (SPD-M20AVP, Shima-
dzu) and a DGU-12A degasser was employed for the analytical
HPLC. A PU-980 pump equipped with a UV-975 detector (JASCO,
Tokyo, Japan) was used for analysis of ethyl linoleate hydroperox-
ides. A LC-6AD recycling system (Shimadzu) equipped with a UV-
970 detector (JASCO) was used for preparative HPLC.
4. HR–ESI-MS (m/z) [MꢀH]ꢀ calcd for C30H39O8S, 559.2366;
found, 559.2387; 1HNMR (400 MHz, CD3OD) d 5.16 (1H, brd,
J = 7.3 Hz, H2), 3.00(1H, brd, J = 13.7 Hz, H3a), 2.99 (1H, dd,
J = 13.7 and 7.3 Hz, H3b), 6.75 (1H, brs, H20), 6.68 (1H, d,
J = 8.2 Hz, H50), 6.65 (1H, brd, J = 8.2 Hz, H6’), 6.33 (1H, d,
J = 16.0 Hz, H200), 8.43 (1H, d, J = 16.0 Hz, H300), 6.83 (1H, d,
J = 8.8 Hz, H5000), 7.23 (1H, d, J = 8.8 Hz, H6000), 2.69 (2H, t,
J = 6.8 Hz, 2000-SCH2), 1.1–1.4 (20H, m), 0.88 (3H, t, J = 6.4 Hz,
CH3); Important correlations observed in the NOESY, H200-H6000,
13
H300-2000-SCH2; CNMR (100 MHz, CD3OD) d 38.2 (C3), 130.7 (C10),
117.5 (C20), 146.0 (C30), 145.2 (C40), 116.3 (C50), 121.9 (C60), 168.5
(C100), 116.7 (C200), 146.1 (C300), 120.0 (C1000), 129.7 (C2000), 148.1
(C3000), 148.6 (C4000), 117.0 (C5000), 123.0 (C6000).
5. HR-ESI-MS (m/z) [MꢀH]ꢀ calcd for C30H39O8S, 559.2366; found,
559.2355; 1HNMR [400 MHz, CD3OD-acetone-d6(2:1, v/v) d 5.16 (1H,
dd, J = 9.6 and 3.4 Hz, H2), 3.41 (1H, dd, J = 14.0 and 3.4 Hz, H3a),
3.19 (1H, dd, J = 14.0 and 9.6 Hz, H3b), 6.64 (1H, s, H20), 6.66 (1H, s,
H50), 6.10 (1H, d, J = 16.0 Hz, H200), 7.39 (1H, d, J = 16.0 Hz, H300), 6.92
(1H, d, J = 1.6 Hz, H2000), 6.67 (1H, d, J = 8.0 Hz, H5000), 6.83 (1H, dd,
J = 8.0 and 1.6 Hz, H2000), 2.68 (2H, t, J = 7.6 Hz, 60-SCH2), 0.78 (3H, t,
J = 7.2 Hz, 60-SC11H22-CH3); 13CNMR (100 MHz, CD3OD) d 37.1 (C3),
122.4 (C10), 121.5 (C20), 147.3 (C30), 145.0 (C40), 115.1 (C50), 133.1
(C60), 168.6 (C100), 115.1 (C200), 147.9 (C300), 127.7 (C1000), 114.8 (C2000),
146.8 (C3000), 149.6 (C4000), 116.4 (C5000), 123.0 (C6000), Signals for C1 and
C2 were not identified for their low intensity.
2.2. HPLC Detection of the antioxidation products from rosmarinic acid
against ethyl linoleate oxidation
Rosmarinic acid (0.4 mg) in CH3CN solution (4 ml) and AIBN
(158 mg) were added to 61 mg of ethyl linoleate in a straight vial
(75 mm height, 40 mm diameter). The control vial was also pre-
pared in a similar manner, but without addition of rosmarinic acid.
Both solutions were well stirred and then incubated at 37 °C for 6 h
with shaking (100 times minꢀ1) by a water bath shaker. Twenty
microlitres of the reaction solution were injected into the analyti-
cal HPLC system under the following conditions, column, Cosmosil
5C18-AR-II (4.6 ꢁ 250 mm, Nacalai Tesque); solvent system, 1%
acetic acid in H2O (solvent A) and CH3CN (solvent B); elution, linear
gradient from 5% of solvent B to 100% of solvent B for 40 min, and
then isocratic mode of 100% solvent B for 10 min; flow rate,
1.0 ml minꢀ1; detection, 280 nm. At intervals for both solutions, a
6. HR-ESI-MS (m/z) [MꢀH]ꢀ calcd for C30H39O8S, 559.2366;
found, 559.2366; 1HNMR (400 MHz, CD3OD) d 5.18 (1H, brd,
J = 9.2 Hz, H2), 3.09 (1H, d, J = 13.6 Hz, H3a), 2.97 (1H, dd, J = 13.6
and 9.2 Hz, H3b), 6.69 (1H, brs, H20), 6.78 (1H, brs, H60), 6.25 (1H,
d, J = 16.0 Hz, H200), 7.54 (1H, d, J = 16.0 Hz, H300), 7.03 (1H, brs,
H2000), 6.76 (1H, d, J = 8.4 Hz, H5000), 6.93 (1H, brd, J = 8.4 Hz, H6000),
2.73 (2H,t, J = 7.6 Hz, 50-SCH2), 1.1–1.5 (20H, m), 0.88 (3H, t,
J = 7.6 Hz, 50-SC11H22-CH3); Important correlations observed in the
NOESY, H60- 50-SCH2; 13CNMR (100 MHz, CD3OD) d 174.5 (C1),
75.3 (C2), 31.8 (C3), 129.7 (C10), 116.7 (C20), 146.0 (C30), 144.8
(C40), 125.4 (C50), 122.4 (C60), 168.5 (C100), 115.2 (C200), 147.5 (C300),
127.7 (C1000), 114.7 (C2000), 146.8 (C3000), 149.7 (C4000), 116.4 (C5000),
123.1 (C6000), 34.8 (50-SCH2), 14.5 (CH3).
20
ll-aliquot was removed from the solution and diluted with
Assignment of 13C signals of 4–6 were based on the reported
data for rosmarinic acid (Kikuzaiki & Nakatani, 1989).
380
ll of methanol. Ten microlitres of the diluted solution was in-
jected into the HPLC to analyse the ethyl linoleate hydroperoxides
using a ODS-A column (4.6 ꢁ 150 mm, YMC, Tokyo, Japan) eluted
with CH3CN/H2O (9:1, v/v) at 1.0 ml minꢀ1; with detection at
234 nm.
2.4. HPLC analysis of the thiol adduct from the antioxidation product of
rosmarinic acid in a lipid oxidation system
2.3. Preparation procedure for thiol adducts (4–6)
One ml of the reaction solution was taken from the antioxidation
vial of rosmarinic acid against ethyl linoleate oxidation to a new vial
1 h after the reaction started. To the new vial was added 1-dode-
To a solution of rosmarinic acid (150 mg) in CH3CN (40 ml) was
added DPPH (2,2-diphenyl-1-picrylhydrazyl, 574 mg) at 23 °C with
stirring. After the reaction mixture was allowed to stand for
10 min, 1-dodecanethiol (253 mg) was added to the mixture. The
mixture was allowed to stand for 1 h at 23 °C and then evaporated
to dryness in vacuo to give a residue (1 g). A part of the residue
(0.4 g) was separated by preparative HPLC under the following
conditions, column, Cosmosil 5C18-AR-II (20 ꢁ 250 mm, Nacalai
canethiol (0.6
(3.5 l) at 23 °C. After standing for 1 h at 23 °C, 20
acid was added to acidify the solution and then 10
l
l) in CH3CN (10
l
l) and subsequently triethylamine
l of phosphoric
l of the solution
l
l
l
was analysed by HPLC under the following conditions: column, Cos-
mosil 5C18-AR-II (4.6 ꢁ 250 mm); solvent system, 1% acetic acid in
H2O (solvent A) and CH3CN (solvent B); elution, linear gradient from
5% of solvent B to 100% of solvent B for 40 min, and then isocratic

A. Fujimoto, T. Masuda / Food Chemistry 132 (2012) 901–906
903
O
O
O
1
O
O
O
O
O
O
O
OH
OH
OH
O
OH
OH
2
2"
5'
3"
OH
2'
2"'
O
OH
O
5"'
HO
HO
OH
OH
1 (RosmarinicAcid)
3
2
O
O
O
O
O
O
O
OH
OH
OH
O
O
S
C12H25
OH
OH
OH
S
C12H25
OH
C12H25
S
HO
OH
OH
HO
HO
OH
OH
OH
5
6
4
Fig. 1. Chemical structures of rosmarinic acid (1), its quinone derivatives (2 and 3), and thiol adducts (4–6). [Numbering system was tentatively used for all compounds
according to a previous publication (Kikuzaiki & Nakatani, 1989)].
mode of 100% solvent B for 10 min; flow rate, 1.0 ml minꢀ1
;
detection, 280 nm. An additional 1 ml solution was taken from the
antioxidation reaction and treated by the same procedure without
addition of 1-decanethiol to obtain the reference HPLC data.
3. Results and discussion
3.1. HPLC analysis of the radical reaction products of rosmarinic acid
The antioxidant activity of rosmarinic acid (1, Fig. 1) and its
antioxidation products were investigated by HPLC. A mixture of
rosmarinic acid (0.25 mM) and ethyl linoleate (25 mM) in acetoni-
trile (4 ml) was oxidised by a radical initiator, AIBN (158 mg), at
37 °C. The oxidation of ethyl linioleate was monitored by the con-
centration of its hydroperoxides using HPLC with detection at
234 nm (Fig. 2).
The antioxidation reaction product produced from rosmarinic
acid was analysed at one-hour intervals by another HPLC under
gradient conditions. Fig. 3 shows that peak 2 was observed at
19.2 min as the major antioxidation product of rosmarinic acid.
The time-course analyses of peaks 1 (rosmarinic acid) and 2 were
carried out, and the data are summarized in Fig. 4. As shown in
Fig. 4, the concentration of rosmarinic acid decreased during the
antioxidation period for 4 h. After 4 h, rosmarinic acid does not
show any antioxidant activity, as shown in Fig. 2. The reaction
product, which was observed as peak 2, first increased, then slowly
decreased up to 4 h (Fig. 4). After 4 h, no typical product could be
observed in the HPLC data (data not shown). These data indicated
that the peak compound 2 should be the initial product produced
by the antioxidation reaction of rosmarinic acid (1) in the lipid oxi-
dation system. Our attempt to isolate compound 2 failed, because 2
was too unstable to be purified by any chromatographic tech-
niques. Direct LC–ESI-MS analysis of peak 2 was, however, success-
ful and the obtained data [m/z 357.0602 (MꢀH)ꢀ] indicated that
the peak 2 compound (2) lost two hydrogens from rosmarinic acid,
revealing that 2 was a quinone derivative of rosmarinic acid.
Fig. 2. Antioxidant activity of rosmarinic acid against AIBN-induced ethyl linoleate
oxidation. [Concentration of reactants: ethyl linoleate (50 mM), AIBN (0.24 M),
rosmarinic acid (0.25 mM or 0 M(control))].
Rosmarinic acid has two catechol sub-structures, one of which is
in the caffeoyl moiety and the other is in the 2-oxyphenylpropa-
noyl moiety. A question arose as to which catechol the quinone
structure was derived from. Antioxidative polyphenols possess
many phenol groups in their structures. For understanding the
antioxidation reaction of the polyphenols, an efficient method to
identify the most reactive position among the many phenol groups
that exist in the polyphenol compounds should be acquired. For
identification of the radical oxidation position of the polyphenols,
Sawai and coworkers (Sawai & Moon, 2000) reported direct NMR
measurements of the polyphenol in the presence of DPPH in a deu-
terium solvent. They revealed quinone formation for the polyphe-
nols by observation of the quinone carbonyl signals in the 13CNMR.
This method is very simple, however, no position information of

904
A. Fujimoto, T. Masuda / Food Chemistry 132 (2012) 901–906
Fig. 3. HPLC analytical data of AIBN-induced oxidation products in ethyl linoleate with rosmarinic acid (1) (reaction time: 1 h); HPLC conditions: column, Cosmosil 5C18-AR-
II (4.6 ꢁ 250 mm); solvent system, 1% acetic acid in H2O (solvent A) and CH3CN (solvent B); elution, linear gradient from 5% of solvent B to 100% of solvent B for 40 min, and
then isocratic mode of 100% solvent B for 10 min; flow rate, 1.0 ml minꢀ1; detection, 280 nm.
should be noted that most thiol compounds have a very unpleasant
smell and this smell has prevented researchers from employing
thiol compounds as a reagent. Recently, Node and coworkers
(Node, 2007) developed several odourless thiol compounds, and
they succeeded in applying them for various syntheses instead of
ordinary thiols. Therefore, we planned to employ one of their
odourless thiols, 1-dodecanethiol, to determine the quinone posi-
tion of the antioxidation product of rosmarinic acid.
3.2. Isolation and structure determination of peak compounds 4–6
DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable radical dehy-
drogenation reagent (Fieser & Fieser, 1967). Hence, we employed
DPPH to produce all the quinone derivatives from rosmarinic acid.
The treatment of rosmarinic acid with ca. 1.5 equivalents of DPPH
in an acetonitrile solution gave two quinone derivatives (2 and 3),
Fig. 4. Time-course analytical data of rosamrinic acid (1) and its antioxidation
product (2) by gradient HPLC.
which were detectable by HPLC at the retention times of 19.1 and
16.5 min, respectively [the retention time of rosmarinic acid
(1):16.7 min] (Fig. 5A). The former corresponded to peak 2 that
the quinone in multi-catechol compounds was obtained. Kondo
and coworkers (Kondo, Kurihara, Miyata, Suzuki, & Toyota, 1999)
reported an antioxidation mechanism of tea catechins based on
an MS fragment consideration and MO calculations. Yoshida et al.
(Yoshida et al., 1989) employed ESR for identification of reacted
phenol in tannins, which are typical multi-catechol compounds
in nature. They observed a radical signal for one of the galloyl
groups in a tannin compound. When the oxidation product of the
antioxidative polyphenol is stable, the chemical structure of the
product would afford important information about the reacted
position in its antioxidation (Masuda, Inaba, & Takeda, 2001;
Krishnamachari, Levine, & Pare, 2002; Wei & Ho, 2006). Chemically,
quinone reacts with nucleophilic substances such as amines and
thiols (Bitter, 2006), and the thiol is the more reactive reagent
(Grant et al., 1986), therefore, several thiol reagents have been
employed for the thiolysis reaction in the structure determination
of some oligomeric biomolecules (Haslam, 1977). In foods, various
thiol adducts to phenolics have also been found (Cejudo-Bastane
et al., 2010). This information indicated that some thiols can be
used to identify the position of the quinone structure that was
produced by the antioxidation reaction of the catechol moiety. It
was observed in the lipid antioxidation reaction of rosmarinic acid.
To the DPPH reaction mixture 1-dodecanethiol (three equivalents)
was added as a nucleophilic and odourless thiol reagent. One hour
later, the HPLC analysis of the reaction mixture revealed that the
two quinone peaks (2 and 3) disappeared and three peaks (4–6,
retention times: 36.0, 36.9, and 37.9, respectively) were produced
(Fig. 5B). These new peak compounds were stable during purifica-
tion, therefore, isolation of the three compounds (4–6) was suc-
cessful as pure forms by a preparative HPLC technique.
Compounds 4–6 were all isolated as colourless oils. Their
molecular formulas were estimated to the same C30H40O8S based
on their HR–ESI-MS results. The MS results indicated that 4–6 were
adducts of one molecule of 1-dodecanethiol to the rosmarinic acid
quinones. The added position of the thiol in each compound was
analysed by NMR. In the 1H-NMR of 4, a proton signal due to the
aromatic ring of the caffeoyl moiety of 4 disappeared compared
to that of rosmarinic acid. This lost proton position should be at
the 2-position of the aromatic ring because the two remaining
proton signals (5- and 6-positions) showed an ortho-coupling rela-
tion (J = 8.2 Hz). These results indicated that the thiol was attached
at the 2000-position, which was supported by two NOE correlations

A. Fujimoto, T. Masuda / Food Chemistry 132 (2012) 901–906
905
Fig. 5. HPLC analytical data of DPPH reaction of rosmarinic acid (1) (panel A) and of 1-dodecanethiol adducts from the DPPH reaction products (panel B); HPLC conditions are
the same as in Fig. 3.
between H200 and H6000, and between H300 and
a-methylene protons
3.3. HPLC analysis of the thiol adduct from the antioxidation product of
rosmarinic acid in lipid oxidation system
of the thiol in the NOESY of 4. In the 1HNMR of 5 and 6, a proton
signal of the aromatic ring of the 2-oxyphenylpropanoyl moiety
of 5 and 6 disappeared from the proton signals of the correspond-
ing aromatic ring of rosmarinic acid, indicating that both 5 and 6
were added products with 1-dodecanethiol on the aromatic ring
of the 2-oxyphenylpropanoyl moiety. The substituted positions of
the thiol were determined to be the 60-position for compound 5
and the 50-position for compound 6 based on the 1D and 2D
NMR analytical results. In the 1HNMR of 5, two sharp singlet pro-
tons were observed in the aromatic ring of the 2-oxyphenylpropa-
noyl moiety, indicating that the two protons existed para to each
other. Therefore, the thiol should be present at the 6-position of
the aromatic ring, which was strongly supported by the up-field
shifted signals (d3.41 and 3.19) of the methylene protons at the
10-position. In the 1HNMR of 6, two singlet protons were also
observed in the aromatic ring of the 2-oxylphenylpropanoyl moi-
ety, however, these singlet signals were broad, thus indicating that
these protons were meta to each other on the aromatic ring. These
results clarified that the thiol existed at the 5-position of the aro-
matic ring, which was supported by an NOE observation between
We succeeded in identifying the structures of the 1-dodecane-
thiol adducts that were produced from the corresponding two qui-
nones of rosmarinic acid (2 and 3). Next, the thiol addition reaction
using the odourless thiol was applied to the AIBN-induced lipid
oxidation system. The addition of 1-dodecanethiol to the quinone
2 did not proceed by the direct addition of the thiol to the antiox-
idation reaction mixture of rosmarinic acid in the lipid oxidation
system. As a disadvantage of 1-dodecanethiol, its reactivity is not
very high compared to the ordinary small molecular thiol com-
pounds. The antioxidation reaction continuously produces radical
species from a radical initiator, AIBN, and the thiol compound
can be easily oxidised under the stated conditions, which probably
prevents the thiol addition to the quinone of rosmarinic acid. After
intensive investigation of the thiol addition conditions, we found
that some organic bases promoted the addition reaction of the
thiol to the quinone. Fig. 6 shows the HPLC analytical results of
the 1-dodecanethiol addition reaction with an excess amount of
triethylamine. The HPLC profile (Fig. 6) clearly shows that the peak
of the quinone 2 disappeared, and the peak of compound 6 was
detected. From the production of 6, the unstable antioxidation
product 2 should be structure 2 in Fig. 1.
H60 (d 6.78) and the
a-methylene protons of the thiol. Based on
these results, compounds 4–6 were determined to be 1-dodecane-
thiol adducts at the 2000-, 50-, and 60-positions, respectively, and the
structures of these isomeric adducts also indicated that compound
4 was derived from the quinone 3, and compounds 5 and 6 were
from the quinone 2 (Fig. 1).
As a conclusion, this investigation revealed that the antioxida-
tion reaction of rosmarinic acid should occur at the catechol posi-
tion of its 2-oxyphenylpropanyl moiety during the first stage of

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the antioxidation against lipid oxidation. Polyphenols possessing
many catechol sub-structures, such as anthocyanins, procyanidins,
and tannins, are well-known as potent complex antioxidants, how-
ever, the first reaction position of most of them during their antiox-
idation has not yet been clarified. This odourless thiol addition
technique should be useful without any unpleasant smell for iden-
tification of the unstable quinone structure as a first reacted posi-
tion of multi-catechol-bearing polyphenols like rosmarinic acid in
this investigation.
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This study was financially supported by The Iijima Memorial
Foundation for The Promotion of Food Science and Technology
(Chiba, Japan), JSPS KAKENHI (No. 23500929) for T. M., and a
Grant-in-Aid for JSPS Fellows (A. F.).
Petersen, M.,
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