Synthesis, characterization and free radical scavenging properties of rosmarinic acid fatty esters

Article abstract of DOI:10.1007/s11746-010-1543-8

The hydrophobation of rosmarinic acid with saturated aliphatic primary alcohols of various chain lengths (methanol to eicosanol) was achieved via an acid-catalyzed esterification in the presence of a highly acidic sulfonic resin. The resulting alkyl rosmarinates were isolated, characterized and their global free radical scavenging activity was determined by the 2,2-diphenyl-1-picrylhy- drazyl method in the stationary state. Only the dodecyl ester showed a stronger activity than rosmarinic acid.

Full text of DOI:10.1007/s11746-010-1543-8

J Am Oil Chem Soc (2010) 87:615–620
DOI 10.1007/s11746-010-1543-8
ORIGINAL PAPER
Synthesis, Characterization and Free Radical Scavenging
Properties of Rosmarinic Acid Fatty Esters
•
Jerome Lecomte Luis Javier Lopez Giraldo
•
´ ˆ
Mickael Laguerre Bruno Barea Pierre Villeneuve
´
•
•
¨
´
Received: 28 July 2009 / Revised: 5 November 2009 / Accepted: 31 December 2009 / Published online: 23 January 2010
Ó AOCS 2010
Abstract The hydrophobation of rosmarinic acid with
saturated aliphatic primary alcohols of various chain
lengths (methanol to eicosanol) was achieved via an acid-
catalyzed esterification in the presence of a highly acidic
sulfonic resin. The resulting alkyl rosmarinates were iso-
lated, characterized and their global free radical scavenging
activity was determined by the 2,2-diphenyl-1-picrylhy-
drazyl method in the stationary state. Only the dodecyl
ester showed a stronger activity than rosmarinic acid.
Lamiaceae family such as rosemary (Rosmarinus offici-
nalis), lemon balm (Melissa officinalis), sage (genus
Salvia), oregano (Origanum vulgare) or savory (genus
Satureja). Several biological activities have been described
for rosmarinic acid, the most known being anti-inflamma-
tory [2] antiviral [3] and antioxidative [4, 5]. Concerning
the later activity, rosmarinic acid as well as its naturally
occurring methyl, ethyl and butyl esters have shown strong
free radical scavenging properties, especially towards
superoxide anion [6]. These properties make them good
candidates to replace some current synthetic antioxidants
(i.e. butylated hydroxytoluene and butylated hydroxyani-
sole) in foods and cosmetics, which are increasingly con-
troversial [7]. However, the implementation of such polar
molecules in lipid-based systems (emulsion or other) is
difficult and can lead to a decrease of their ability to
counteract oxidation of unsaturated lipids at the oil–water
interface [8, 9]. To solve this issue, one strategy consists of
adjusting their polarity by the grafting of aliphatic chains of
increasing length [10, 11]. Herein, we reported for the first
time the synthesis and characterization (1H-13C-NMR and
ESI-MS) of alkyl esters of rosmarinic acid with carbon
chain lengths ranging from 1 to 20 carbon atoms (com-
pounds R1–R20), and the evaluation of their global free
radical scavenging activity by the well known 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) method.
Keywords Rosmarinic acid fatty esters ꢀ Syntheses ꢀ
NMR and mass spectrometry characterization ꢀ Radical
scavenging properties ꢀ DPPH ꢀ Stationary state
Introduction
Rosmarinic acid (RO) [(R)-a-[[3-(3,4-dihydroxyphenyl)-1-
oxo-2E-propenyl]oxy]-3,4-dihydroxy-benzenepropanoic
acid] is a polyphenolic compound present in high amount
(C3% w/w dry matter) in many herbal plants [1] of the
´
J. Lecomte (&) ꢀ M. Laguerre ꢀ B. Barea ꢀ P. Villeneuve
CIRAD, UMR IATE, 34398 Montpellier, France
e-mail: jerome.lecomte@cirad.fr
P. Villeneuve
e-mail: pierre.villeneuve@cirad.fr
M. Laguerre
Materials and Methods
Department of Food Science, Chenoweth Laboratory,
University of Massachusetts, 100 Holdsworth Way,
Amherst, MA 01003, USA
Materials
L. J. L. Giraldo
Ò
˚
Sulfonic resin Amberlite IR-120H, molecular sieve (3 A),
Department of Chemical Engineering,
Industrial University of Santander,
Bucaramanga, Colombia
rosmarinic acid (96.5%), 2,2-diphenyl-1-picrylhydrazyl,
n-butanol (99.8%), n-octanol (99%), n-dodecanol (99.5%),
123

616
J Am Oil Chem Soc (2010) 87:615–620
n-hexadecanol (99%), n-octadecanol (99%), n-eicosanol
(98%) and solvents (analytical or HPLC grade) were pur-
chased from Sigma-Aldrich (Saint Quentin, France). Silica
back to 0/100 (v/v) in 5 min. Rosmarinic acid (R0) and its
esters (R1–R20) were detected under UV light at 328 nm
and their retention time, in minutes, were (R0): 19.6, (R1):
21.4, (R4): 25.4, (R8): 29.6, (R12): 32.5, (R16): 34.4,
(R18): 35.5 and (R20): 36.7 . As the alkyl rosmarinate
were the only product of the reaction between rosmarinic
acid and the alcohol, the molar yield of the ester formed
was defined as follows:
˚
(60–200 lm, pore size 60 A) was from Acros organics
(Geel, Belgium). Available immobilized lipase from
C. Antarctica lipase B (NovozymÒ 435) was purchased
from Novozymes A/S (Bagsvaerd, Denmark).
Methods
Molar yield ð%Þ of ester ¼ 100ðmol of esterÞ=
ðmol of ester þ mol of remaining R0Þ:
Chemical Synthesis and Purification of Alkyl Rosmarinates
It was calculated from the calibration curves established
from purified esters. The reaction was stopped when the
molar yield of the ester was at a maximum.
The chemical esterification of rosmarinic acid (56 lmol)
was carried out in sealed flasks each containing 5 mL of
alcohol (methanol, 123.44 mmol; n-butanol, 54.64 mmol;
n-octanol, 31.905 mmol; n-dodecanol, 22.46 mmol; n-hex-
adecanol, 16.95 mmol; n-octadecanol, 15.09 mmol and
n-eicosanol, 13.6 mmol). The reaction mixtures were stirred
(orbital shaker, 250 rpm, 55–70 °C) prior to the addition of
the catalyst, the strongly acidic sulfonic resin AmberliteÒ
IR-120H (5% w/w—total weight of both substrates) previ-
ously dried at 110 °C for 48 h. The water generated during
the reaction was removed by absorption on molecular sieves
(40 mg/mL) added to the medium. Samples (20 lL) were
regularly withdrawn from the reaction medium then mixed
with 980 lL of methanol, filtered (0.45 lm syringe filter
MillexÒ-FH, Millipore Corporation Bedford, MA, USA)
and finally analyzed by reverse phase HPLC with UV
detection at 328 nm. After complete (4–21 days) conversion
of rosmarinic acid into the corresponding ester the latter was
purified in a two-step procedure. Firstly, a liquid–liquid
extraction using hexane and acetonitrile was achieved to
remove the excess of alcohol. Then, the remaining traces of
the alcohol and rosmarinic acid were eliminated by flash
chromatography on a CombiFlashÒ CompanionÒ system
(Teledyne Isco Inc., Lincoln, NE, USA). Separation was
achieved on a silica column using an elution gradient of
hexane and ether (20–100% in 35 min). The purity of the
esters, obtained as pale yellow to yellow amorphous pow-
ders, was checked by HPLC and the absence of residual
alcohol was confirmed by TLC.
1
ESI-MS, H-NMR and 13C-NMR Characterization
Pure esters were first characterized by electrospray ionisa-
tion-mass spectrometry (ESI-MS) in negative mode using a
mass spectrometer composed of a quadrupole ion trap mass
analyzer, an external atmospheric pressure ion source and
an integrated syringe pump (Finnigan LCQ Serie MS
detector, San Jose, CA, USA). Methanolic solutions of
esters (100 mg/L) were directly injected into the ESI-source
(infusion mode) at a 5-lL/min flow rate. The zoom scan
mode was used for an accurate determination of the parent
ion m/z, while fragment ions were obtained in MS2 mode.
Basic operating conditions in the negative mode were the
following: collision energy of 30–35%, ionization voltage
of 4.5 kV, capillary temperature of 300 °C and a sheath gas
(nitrogen) flow of 20 arbitrary units.
1H- and 13C-NMR spectra were recorded in DMSO with
a Bruker DRX 400 spectrometer at 400 and 100 MHz
respectively. In the case of alkyl rosmarinates represented
as RCO–O–aCH2–bCH2–(CH2)n-3–CH3, three types of
methylene were distinguished in the alkyl chain for the
attribution of chemical shifts: –aCH2– and –bCH2– corre-
spond to methylene in alpha- and beta-position related to
oxygen respectively, and –(CH2)n-3– correspond to the
other methylene groups, where n is the total number of
carbon of the alkyl chain.
HPLC Monitoring of Rosmarinic Acid Conversion
Radical Scavenging Activity Assessment by the DPPH
Method and Determination of Stationary Parameters
Monitoring of ester formation was carried out with a ACEÒ
5 C18 reversed phase column (5 lm, 250 9 4.6 mm,
˚
100 A) (ACT, Aberdeen, Scotland) using a Dionex
Basically, the DPPH method consists in measuring the
ability of a molecule to reduce the 2,2-diphenyl-1-picryl-
hydrazyl radical (DPPHꢀ) in methanol and its subsequent
bleaching at 515 nm. The radical scavenging activity of
rosmarinic acid and its esters against DPPHꢀ was performed
spectrometrically at 515 nm and 20 °C, as described by
Brand-Williams et al. [12] and recently applied to
Ultimate 3000 HPLC system (Dionex, Jouy En Josas,
France). Peak integration was performed using Chrome-
leon software (Version 6.8). Briefly, gradient elution was
performed using methanol and phosphoric acid 3 mM at
1 mL/min and 25 °C, in linear gradients from 0/100 (v/v)
to 100/0 (v/v) for 30 min, then 100/0 (v/v) for 10 min and
123

J Am Oil Chem Soc (2010) 87:615–620
617
´
chlorogenate fatty esters by Lopez Giraldo et al. [13].
(R1): 98.5%, (R4): 99.3%, (R8): 99.5%, (R12): 94.4%,
(R16): 81.6%, (R18): 99.0% and (R20): 99.0%. It corre-
sponded to the quasi complete conversion of rosmarinic
acid, except for hexadecyl rosmarinate where the maxi-
mum acid conversion was 82%. After the purification
steps, the purity of esters was at least 96% and only traces
of rosmarinic acid were detected by HPLC. The remaining
4% were attributed to the impurities already present in the
starting material (rosmarinic acid, 96.5% purity) or to
their derivatives, that could not be eliminated by flash
chromatography.
Briefly, the absorbance decay was monitored on solutions
consisting in 1,950 lL methanolic DPPHꢀ solution
(60 lM) and 50 lL methanolic solution of phenolic com-
pound (14.6–585 lM). The absorbance decay was moni-
tored each minute until it reached a steady state (14 h for
rosmarinic acid and 6 h for its esters) that corresponds to
the complete consumption of the antioxidant. In absence of
antioxidant (blank), the spontaneous DPPHꢀ bleaching
during the experiment appeared to be low and did not
exceed 3–4% and 6–7% after 6 and 14 h respectively.
However, a correction of this intrinsic DPPHꢀ bleaching
was systematically made in order to avoid any bias. The
global radical scavenging activity was defined as the total
number of reduced DPPHꢀ at stationary state (nss) and
expressed as a mol DPPHꢀ radical reduced by a mol of
tested compound. It was calculated as 1/(2 9 EC50) where
EC50 corresponds to the amount of antioxidant necessary to
reduce the initial DPPHꢀ concentration by 50%. All deter-
minations were done in triplicate.
NMR and MS Characterization of Alkyl Rosmarinates
Rosmarinic acid and its esters were first characterized by
1H NMR and 13C NMR (Table 1). Data obtained for ros-
marinic acid, and its methyl and butyl esters were highly
consistent with those reported in the literature [14–16]. For
1
alkyl chains of more than four carbon atoms, H NMR of
–aCH2–, –bCH2– and other methylene group give multi-
plets with chemical shifts of 4.07–4.03, 1.54–1.51 and
1.37–1.23 ppm respectively. On the other hand, 13C-NMR
chemical shifts of the same methylene were respectively
64.6–64.3, 31.3–31.1 and 29.3–22.1 ppm.
Results and Discussion
Synthesis of Alkyl Ester of Rosmarinic Acid
ESI-MS spectra (full scan mode) of both rosmarinic
acid and its esters (Table 2) showed two peaks corre-
sponding to the molecular ion of the deprotonated com-
pound [MM–H] and its adduct [2MM–H]. The MS2
fragmentation of rosmarinic acid molecular ion (358.97)
lead to three peaks at 197, 179 and 161 (m/z) corre-
sponding to the deprotonated form of 3-(3,4-dihydroxy-
phenyl)lactic and caffeic acids and their dehydrated
forms, respectively (Fig. 2; Table 2). These results were
in agreement with the fragmentation scheme proposed by
Møller et al. [17].
In a first attempt, rosmarinic acid or its methyl ester were
enzymatically esterified following a procedure already
used in a previous work [10] for the esterification of
chlorogenic acid (5-caffeoyl quinic acid, 5-CQA). How-
ever, none of the corresponding esters was obtained
whatever the alcohol used and the reaction conditions. The
reason for such a failure was attributed to the putative
inhibition of the enzyme due to steric hindrance at the
catalytic site. Then, alkyl rosmarinates were obtained by
acid catalyzed esterification of rosmarinic acid with the
corresponding saturated aliphatic primary alcohols (Fig. 1)
in the presence of a highly acidic sulfonic resin. The
reaction was achieved at low temperature (55–70 °C) to
avoid rosmarinic degradation, in an excess of the alcohol
that played, when melted, both the role of substrate and
solvent. The reaction was stopped after 4–21 days, when
the molar yield of the ester has reached its maximum:
In case of alkyl rosmarinates, fragmentation of
molecular ion always leads to only two peaks at 179
and 135 (m/z) whose correspond to the deprotonated form
of caffeic acid and its residue after decarboxylation,
respectively (Fig. 3; Table 2). The differences in frag-
mentation between rosmarinic acid and its esters may be
due to the higher weakness of the ester bond at carbon 9
compared to carbon 90.
5'
5'
9'
OH
OH
9'
OH
OH
6'
6'
O
CO2R
O
CO2H
7
7
R-OH
2
2
HO
HO
HO
HO
1
1
9
8'
9
8'
1'
1'
O
O
Amberlite IR120
7'
7'
2'
2'
8
8
55-70°C, solvent free
6
6
R = CnH2n+1
5
5
R0
R1 : n=1
R16 : n=16
R4 : n=4
R18 : n=18
R8 : n=8
R20 : n=20
R12 : n=12
Fig. 1 Heterogeneous acid-catalyzed esterification of rosmarinic acid with aliphatic alcohols of various chain length
123

618
J Am Oil Chem Soc (2010) 87:615–620
1
Table 1 13C-NMR (100 MHz, DMSO) and H-NMR (400 MHz, DMSO) data for rosmarinic acid (R0) and its esters (R1–R20)
1
(d/ppm) H
C
(d/ppm) 13C
R0
R1–R20
R0
R1–R20
1
125.3 125.3
113.2 112.8
144.9 145.0
148.6 148.7
115.7 115.7
121.6 121.6
145.9 146.2
114.8 114.9
165.9 165.9
127.3 126.6
116.7 116.6
145.6 145.6
144.0 144.1
115.4 115.3
120.0 120.0
36.1 36.2
–
–
–
–
–
sa
2
1H 7.08
s
7.07
3
–
–
–
–
–
–
–
4
–
–
–
5
1H 6.80–6.78
1H 7.04–7.02
1H 7.51–7.47
1H 6.28–6.24
d, J = 9.2 Hz
6.79–6.77
7.02–7.00
7.52–7.48
6.28–6.24
–
da, J = 8.1 Hz
6
d, J = 8.5 Hz
d, J = 8.3 Hz
7
d, Jtrans = 16.9 Hz
d, Jtrans = 15.8 Hz
8
d, Jtrans = 16.2 Hz
d, Jtrans = 15.9 Hz
9
10
20
30
40
50
60
70
–
–
–
–
–
–
–
–
–
1H 6.70–6.69
d, J = 2.0 Hz
6.68–6.67
–
d, J = 2.0 Hz
–
–
–
–
–
–
–
–
–
1H 6.67–6.65
1H 6.56–6.54
d, J = 9.3
6.66–6.64
6.52–6.50
d, J = 8.0 Hz
dd, J = 8.1, 2.1 Hz
dd, J = 8.1, 1.9 Hz
ma
2H 3.04–2.99, 2.96–2.90 dd, J = 14.9, 4.5 Hz dd, J = 14.5, 8.7 Hz 2.98–2.96
80
90
72.8 72.9
1H 5.07–5.04
dd, J = 8.3, 4.6 Hz
5.12–5.08
–
dd, J = 7.2, 5.7 Hz
170.9 169.4
–
–
–
–
–
–
–
–
–
–
–
–
–aCH2–
–bCH2–
–CH2–
–CH3
64.6–64.3
2H
2H
xHc
4.07–4.03
1.54–1.51
1.37–1.23
m
31.3–31.1
29.3–22.1
13.91 (51.96b) 3H
m
m
m (sb)
0.90–0.82
(3.69b)
a
s singlet, d doublet, m multiplet, dd doublet of doublet
b
c
Data for methyl rosmarinate (–OCH3)
x = 2(n-3) with n total number of carbon in the alkyl chain
Table 2 Negative ion and MS2
data for rosmarinic acid (R0)
and some of its esters
Compound (Id. no.)
MM (g mol-1
)
Full scan (m/z) [MM–H]a
MS2
Rosmarinic acid
360.31
358.97 (100)
718.90 (72)
161.04 (100)
179.02 (26)
197.01 (22)
135.12 (26)
178.96 (100)
135.14 (28)
178.97 (100)
135.11 (23)
178.99 (100)
135.09 (27)
178.95 (100)
ND
(R0)
Methyl rosmarinate
(R1)
374.34
416.42
528.63
612.79
640.85
373.04 (100)
786.84 (58)
415.11 (26)
831.21 (100)
527.25 (52)
1,055.21 (100)
611.36 (62)
1,223.36 (100)
639.41 (58)
1,279.42 (100)
Butyl rosmarinate
(R4)
Dodecyl rosmarinate
(R12)
Octadecyl rosmarinate
(R18)
MM molar mass, ND not
determined
a
Value into brackets
corresponds to percentage
relative intensity
Eicosyl rosmarinate
(R20)
As a consequence, the fragmentation of molecular ion of
each ester theoretically led to the breaking of this weaker
ester bond into the deprotonated form of caffeic acid and
3-(3,4-dihydroxyphenyl) lactates. However, the fragment
containing the alkyl chain was not detected under our
working conditions.
123

J Am Oil Chem Soc (2010) 87:615–620
619
5'
2'
9'
OH
OH
antioxidant at stationary state. Surprisingly, it appeared
that only dodecyl rosmarinate (R12) with a nss value of
12.36 0.19 could reduce more DPPHꢀ than rosmarinic
acid (nss = 9.78 0.06).
6'
O
CO2H
7
2
HO
HO
1
9
8'
m/z = 359
1'
O
7'
8
6
5
Indeed, in a similar work on chlorogenic acid (5-CQA)
and its alkyl esters [13] it was found that all chlorogenates
exhibit higher nss values (6.2–8.5) than 5-CQA (4.9).
However, it is worth mentioning that rosmarinic acid
reached a steady state in 14 h, whereas all esters reached it
in 6 h. Interestingly, such a strong influence of the esteri-
fication on the DPPHꢀ bleaching was already observed with
chlorogenic acid and its alkyl ester [13] and protocatechuic
acid and its methyl ester [18]. In addition, it can be
observed that all nss values were greater than 4 which is the
number of available phenolic hydroxyl groups present in
each tested molecule and known as being responsible for
the antiradical activity. These results suggest, as proposed
by other authors [19] the possible nucleophilic methanol
regeneration of oxidized forms (quinone) of antioxidant
into new reducing phenolic compounds and its combina-
tion with dimerization phenomena. In the future, further
investigations on structure of reaction products will be
required to determine the part of each mechanism involved
according to alkyl chain length.
O
7
2
5'
2'
HO
9'
OH
1
6
9
6'
CO2-
O
8
8'
1'
HO
HO
OH
7'
5
m/z = 179
m/z = 197
Dehydrated ion
fragments (m/z = 161)
-H2O
-2H2O
Fig. 2 MS2 fragmentation scheme of rosmarinic acid as proposed by
Møller et al. [17]
5'
9'
OH
OH
6'
O
CO2R
7
2
HO
HO
1
9
8'
1'
O
7'
2'
8
6
5
O
7
7
2
5
2
HO
HO
HO
In conclusion, this work relates the first heterogeneous
synthesis of a homologous series of alkyl rosmarinates of
chain lengths ranging from 1 (methyl) to 20 (eicosyl)
carbon atoms and their full characterization. In terms of
global radical scavenging properties according to the
DPPH method at the stationary sate, it shows that the
grafting of an alkyl chain to rosmarinic acid does not
necessarily lead to an increase of its stoichiometry against
DPPHꢀ (except for dodecyl), despite the drastic accelera-
tion of the free radical scavenging process.
1
1
9
O
8
8
-CO2
6
6
m/z = 179
m/z = 135
HO
5
Fig. 3 MS2 fragmentation scheme of alkyl rosmarinates
Table 3 Global radical scavenging capacity (nss) of rosmarinic acid
(R0) and its esters (R1–R20) determined at the stationary state
nss
0
Compound
Alkyl chain (–OC9 –OR)
R0
R=H
9.78 0.06
R1
R=CH3 (methyl)
9.22 0.07
8.61 0.12
6.24 0.02
12.36 0.19
9.22 0.07
6.82 0.28
6.35 0.08
´
Acknowledgments Luis Javier Lopez Giraldo thanks the support
R4
R=(CH2)3CH3 (butyl)
R=(CH2)7CH3 (octyl)
R=(CH2)11CH3 (dodecyl)
R=(CH2)15CH3 (hexadecyl)
R=(CH2)17CH3 (octadecyl)
R=(CH2)19CH3 (eicosyl)
from Programme Alban, the European Union Programme of High Level
Scholarships for Latin America, scholarship N°. E05D055786CO.
R8
R12
R16
R18
R20
References
1. Lamaison JL, Petitjean-Freytet C, Carnat A (1991) Medicinal
Lamiaceae with antioxidant properties, a potential source of
rosmarinic acid. Pharm Acta Helv 66:185–188
nss: mol DPPH radical reduced by mol of compound, at the stationary
state
Data are means of triplicate values
2. Osakabe N, Takano H, Sanbongi C, Yasuda A, Yanagisawa R,
Inoue K, Yoshikawa T (2004) Anti-inflammatory and anti-allergic
effect of rosmarinic acid (RA); inhibition of seasonal allergic rhi-
noconjunctivitis (SAR) and its mechanism. Biofactors 21:127–131
3. Swarup V, Ghosh J, Ghosh S, Saxena A, Basu A (2007) Antiviral
and anti-inflammatory effects of rosmarinic acid in an experi-
mental murine model of Japanese encephalitis. Antimicrob
Agents Chemother 51:3367–3370
Radical Scavenging Properties of Alkyl Rosmarinates
In terms of antioxidant properties, we reported in Table 3
the global free radical scavenging activity (nss) of ros-
marinic acid and its ester determined by the DPPH
method. The global free radical scavenging activity was
defined as the total mole of DPPH reduced by mole of
´
´
4. Laguerre M, Lopez-Giraldo LJ, Lecomte J, Barea B, Cambon E,
Tchobo PF, Barouh N, Villeneuve P (2008) Conjugated autoxi-
dizable triene (CAT) assay: a novel spectrophotometric method
123

620
J Am Oil Chem Soc (2010) 87:615–620
´
13. Lopez Giraldo LJ, Laguerre M, Lecomte J, Figueroa-Espinoza
´
for determination of antioxidant capacity using triacylglycerol as
ultraviolet probe. Anal Biochem 380:282–290
MC, Barea B, Weiss J, Decker EA, Villeneuve P (2009) Kinetic
5. Exarchou V, Nenadis N, Tsimidou M, Gerothanassis IP, Troganis
A, Boskou D (2002) Antioxidant activities and phenolic com-
position of extracts from Greek oregano, Greek sage, and summer
savory. J Agric Food Chem 50:5294–5299
and stoichiometry of the reaction of chlorogenic acid and its alkyl
esters against the DPPH radical. J Agric Food Chem 57:863–870
14. Lu Y, Foo LY (1999) Rosmarinic acid derivatives from Salvia
officinalis. Phytochemistry 51:91–94
6. Choudhary MI, Begum A, Abbaskhan A, Ajaz A, Shafique-
Ur-Rehman, Atta-Ur-Rahman (2005) Phenyl polypropanoids
from Lindelofia stylosa. Chem Pharm Bull 53:1469–1471
7. Iverson F (1995) Phenolic antioxidants: health protection branch
studies on butylated hydroxyanisole. Cancer Lett 93:49–54
8. Decker EA, Warner K, Richards MP, Shahidi F (2005) Measuring
antioxidant effectiveness in food. J Agric Food Chem 53:4303–
4310
15. Fecka I, Turek S (2008) Determination of polyphenolic com-
pounds in commercial herbal drugs and spices from Lamiaceae:
thyme, wild thyme and sweet marjoram by chromatographic
techniques. Food Chem 108:1039–1053
16. Huang H, Chao QR, Tan RX, Sun HD, Wang DC, Ma J, Zhao SX
(1999) A new rosmarinic acid derivative from Isodon oresbius.
Plant Med 65:92–93
17. Møller JKS, Catharino RR, Eberlin MN (2007) Electrospray
ionization mass spectrometry fingerprinting of essential oils:
spices from the Labiatae family. Food Chem 100:1283–1288
18. Saito S, Kawabata J (2006) DPPH (=2, 2-diphenyl-1-picrylhy-
drazyl) radical-scavenging reaction of protocatechuic acid (=3,
4-dihydroxybenzoic acid): difference in reactivity between acids
and their esters. Helv Chim Acta 89:1395–1407
19. Saito S, Kawabata J (2005) Effects of electron-withdrawing
substituents on DPPH radical scavenging reactions of proto-
catechuic acid and its analogues in alcoholic solvents. Tetrahedron
61:8101–8108
9. Frankel EN, Meyer AS (2000) The problems of using one-
dimensional methods to evaluate multifunctional food and bio-
logical antioxidants. J Sci Food Agric 80:1925–1941
´
10. Lopez Giraldo LJ, Laguerre M, Lecomte J, Figueroa-Espinoza
MC, Barouh N, Barea B, Villeneuve P (2007) Lipase- catalyzed
´
synthesis of chlorogenate fatty esters in solvent-free medium.
Enzyme Microb Technol 41:721–726
11. Figueroa-Espinoza MC, Villeneuve P (2005) Phenolic acids
enzymatic lipophilization. J Agric Food Chem 53:2779–2787
12. Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free
radical method to evaluate antioxidant activity. Lebensm Wiss
Technol 28:25–30
123