Antioxidant potential of hydroxycinnamic acid glycoside esters

Article abstract of DOI:10.1021/jf800317v

Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals. In this study, the antioxidant activity of various types of hydroxycinnamoyl glycoside esters that mimic the structure of polymeric carbohydrates was studied in diffe

Full text of DOI:10.1021/jf800317v

J. Agric. Food Chem. 2008, 56, 4797–4805 4797
Antioxidant Potential of Hydroxycinnamic Acid
Glycoside Esters
PETRI KYLLI,*,† PAULA NOUSIAINEN,‡ PETER BIELY,§ JUSSI SIPILÄ,‡
†
MAIJA TENKANEN,† AND MARINA HEINONEN
Department of Applied Chemistry and Microbiology, P.O. Box 27, 00014 University of Helsinki,
Finland; Laboratory of Organic Chemistry, Department of Chemistry, P.O. Box 55, 00014 University
of Helsinki, Finland; and Institute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9,
845 38 Bratislava, Slovakia
Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals. In this study,
the antioxidant activity of various types of hydroxycinnamoyl glycoside esters that mimic the structure
of polymeric carbohydrates was studied in different model systems prone to oxidation, namely,
liposomes and emulsions. In addition, radical scavenging activity against the stable 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical was tested. It was found that the esterification in the primary hydroxyl
group of the glycoside resulted in the improved radical scavenging activity of both sinapoyl and feruloyl
glycosides compared to conjugation to the secondary hydroxyl group. Increased activity was also
observed, particularly in the case of feruloyl glucosides in inhibiting the oxidation of liposomes
emulsions. The results showed that sinapic and ferulic acid glycoside esters were as effective or
more efficient antioxidants than their free forms. In conclusion, the strength of their antioxidant effect
depends on the nature of conjugation.
KEYWORDS: Hydroxycinnamic acid; ferulic acid; sinapic acid; glycoside ester; conjugation; radical
scavenging; antioxidant activity
INTRODUCTION
and p-coumaric acid, which are found in the different parts of
grains such as wheat bran, maize bran, and rice endosperm (12).
Among the different hydroxycinnamic acids, ferulic acid has
special importance. Ferulic acid is esterified to various cell wall
polysaccharides. It is linked at position C-5 to R-L-arabinofura-
nosyl side groups in arabinoxylans, at position C-2 to R-L-
arabinofuranosyl residues in arabinans, at position C-6 to ꢀ-D-
galactopyranosyl residues in galactans (11), and at position C-4
to R-D-xylopyranosyl side groups in xyloglucans (9). Ferulic
acid has a significant role in cross-linking cell wall polysac-
charides (13), and cereals contain moderate levels of ferulic acid
dehydrodimers, which are formed by oxidative coupling
reactions (4, 8).
Phenolic acids are considered to be anti-inflammatory,
anticarcinogenic,andantimicrobialagentsaswellasantioxidants(14–16).
Benefits provided by phenolic acids are assumed to be due to
their antioxidant activity: metal ion chelating, radical scavenging,
and inhibition of prooxidant enzymes (17). The antiradical
activity of polyphenols is ascribed to the hydroxyl groups and
the availability of phenolic hydrogen for donation (2, 15).
Hydroxycinnamic acids have been shown to exhibit singlet
oxygen quenching. The electron-donating ability of the substit-
uents and the polarity of the solvent influence the quenching
rate. Charge transfer is thereby attended to singlet oxygen
quenching (18). Phenolic compounds having one hydroxyl group
on their aromatic ring are less effective antioxidants than
phenolics with the second hydroxyl in the ortho or para position.
Hydroxycinnamic acid derivatives have been found in several
fruits, vegetables, and cereals, in which they usually appear as
the esters of quinic acid, sterol, glucose, or plant cell wall
polysaccharides (1–4). Raspberry, coffee, and crispbread are
among the best dietary sources of phenolic acids (5), with large
amounts of phenolic acids also present in rye (6–8). In various
fruits and vegetables the main phenolic acid component is
chlorogenic acids such as 3-caffeoylquinic acid, 4-caffeoylquinic
acid, or 5-caffeoylquinic acid, but other phenolic acids are also
found. p-Coumaric acid, ferulic acid, sinapic acid, 5-hydroxy-
ferulic acid, and dehydrodimers of ferulic and sinapic acids have
been isolated from the hydrolysates of plant cell walls (3, 4, 9).
Winter and Herrmann (1) identified p-coumaroyl-, caffeoyl-,
and feruloyl-glucosides esterified to the C-1 hydroxyl group
from tomatoes, bell peppers, and eggplants. In corn and barley,
cell walls have been found to contain esterified p-coumaric and
ferulic acid and also some 5-hydroxyferulic acid and sinapic
acid (10). In cereals, the main phenolic acids are ferulic acid
* Author to whom correspondence should be addressed (telephone
+358
9
191 58281; fax +358
9
191 58475; e-mail
petri.kylli@helsinki.fi).
† Department of Applied Chemistry and Microbiology, University
of Helsinki.
‡ Department of Chemistry, University of Helsinki.
§ Slovak Academy of Sciences.
10.1021/jf800317v CCC: $40.75  2008 American Chemical Society
Published on Web 05/22/2008

4798 J. Agric. Food Chem., Vol. 56, No. 12, 2008
Kylli et al.
Figure 1. Hydroxycinnamic acid ester conjugates of methyl glucose.
Scheme 1. Synthesis of Methyl 6-O-Feruloyl-glucopyranoside Ester (26)
Denmark). Emultop emulgator was from Degussa Texturants Systems
Deutschland GmbH & Co. KG (Hamburg, Germany). Tocopherols were
removed from rapeseed oil (Kultasula, Mildola, Finland) prior to use
as described by Lampi et al. (22). 4-Nitrophenyl ferulate was a gift
from Dr. M. Mastihubova´ (Institute of Chemistry, Slovak Aacdemy of
Sciences, Bratislava, Slovakia).
However, the presence of more than three hydroxyl groups on
an aromatic ring does not improve the efficacy. Kikuzaki et al.
(19) showed that alkyl esters of ferulic acid and gallic acid have
a lower antioxidant efficacy than free acid in bulk methyl
linoleate at 40 °C. Alkyl esters of ferulic acid were, however,
more active than ferulic acid in ethanolic buffer. Ferulic acid
dehydrodimers have been shown in vitro to be more effective
antioxidants than monomeric ferulic acid in both the aqueous
and lipid phases (20, 21).
Free hydroxycinnamic acids are widely examined, and they
are known to have antioxidant potential, but the antioxidant
activity of conjugated hydroxycinnamates is less investigated.
The aim of this paper was to evaluate the antioxidant activity
of various glycoside esters and to assess whether the nature of
ester conjugation has an effect on the antioxidant activity. It
was hypothesized that phenolic acids esterified to the primary
hydroxyl group of the glycoside could be more effective
antioxidants compared to other types of conjugates. To mimic
the polymeric structure of carbohydrates, phenolic acids were
esterified to primary hydroxyl groups (C-5 or C-6) and to
secondary hydroxyl groups (C-2, C-3, and C-4), whereas the
naturally favored position C-1 of monosaccharides was blocked
by methyl or 4-nitrophenyl groups.
Synthesis of Hydroxycinnamic Acid Glucoside Ester Conjugates.
Ferulic acid and sinapic acid ester conjugates of methyl glucoside
(Figure 1) were synthesized from methyl R-D-glucopyranose and trans-
hydroxycinnamic acids as presented in Scheme 1. Position C1 in
glycosides was blocked by methylation to prevent the esterification to
that position to mimic the natural conditions in polymeric carbohydrates
where the position C1 is not available. Ester conjugates were prepared
in two fashions: using “batch” method to obtain isomeric monoester
conjugate mixtures and, on the other hand, by synthesizing specific
regioisomers of particular ester conjugates of interest (24–27).
Synthesis of Ferulic Acid DeriVatiVes. Dehydrodimer of ferulic acid
(Figure 2) was synthesized according to the method by Ralph et al.
(28), and for the synthesis of 5-hydroxyferulic acid the methods outlined
by Ellis and Lenger (29) and Lam et al. (30) were followed.
Synthesis of 4-Nitrophenyl Feruloyl Arabinofuranoside and Xylopy-
ranosides. 4-Nitrophenyl glycoside conjugates contained another type
of sugar moiety. They were used for the evaluation of the effect of
addition of sugar to the hydroxycinnamic acid. 4-Nitrophenyl 2-O-
MATERIALS AND METHODS
Materials. Ferulic acid, pyrogallol, and 1,1-diphenyl-2-picrylhy-
drazyl (DPPH) reagent were from Extrasynthe`se (Genay, France). All
solvents were of HPLC grade and purchased from Rathburn Chemicals
Ltd. (Walkerburn, Scotland). A Milli-Q water purification system was
used (Millipore, Bedford, MA). Ammonium dihydrogen phosphate,
sodium dihydrogen phosphate, disodium hydrogen phosphate cupric
acetate, copper sulfate, sodium chloride, and R-tocopherol were from
Merck (Darmstadt, Germany). Lecithin from soybean (containing 40%
phosphatidylcholine) and sinapic acid were from Sigma Chemical Co.
(St. Louis, MO). Ultraflo L was provided by Novozymes (Bagsvaerd,
Figure 2. Structures of 5-hydroxyferulic acid and ferulic acid
dehydrodimer.

Hydoxycinnamic Acid Glycoside Esters
J. Agric. Food Chem., Vol. 56, No. 12, 2008 4799
photometer (Norwalk, CT) (33). Ferulic acid and sinapic acid were
used as standard compounds.
Antioxidant Activity Testing. Antioxidant activities were tested by
using three different models. The DPPH radical scavenging test was
used to evaluate the theoretical antioxidant potential of hydroxycin-
namoyl glycoside esters. Liposome and emulsion lipid oxidation models
represent two different structures naturally occurring in foods. Tested
compounds solubilized in methanol were dosed on the basis of the total
phenolic content.
DPPH Radical ScaVenging Test. The test was performed according
to the method of Ka¨hko¨nen et al. (34). Methanolic 0.1 mM DPPH•
radical solution (2.950 mL) was mixed with 0.050 mL of the tested
compound. The concentration of the sample was 1 mg/mL based on
the content of total phenolics. All samples were dissolved in methanol.
Molar concentrations of different hydroxycinnamic glycosides varied
from 4.5 to 12.4 mM, but there was no significant difference in the
molar concentration between similar types of hydroxycinnamates. The
absorption was measured at 517 nm every 30 s for 4 min. The results
were expressed as the percentage of radicals scavenged in 4 min of
reaction time. The percentage of radical scavenging activity was
calculated as
radical scavenging activity (%) ) 100 × (A1 - A2)/A1
where A1 ) the initial absorbance at beginning of the reaction (t ) 0)
and A2 ) the absorbance after 4 min of reaction time. DPPH• radical
scavenging activity test was performed in triplicate measurements using
pyrogallol (0.5 mg/mL) as a control sample.
Figure 3. Structures of 4-nitrophenyl 2-O-feruloyl- and 4-O-feruloyl-ꢀ-D-
xylopyranosides, 5-O-feruloyl-R-L-arabinofuranoside, and 4-nitrophenyl
ferulate.
Emulsion Model System. The emulsion (10% o/w) oxidation model
system (34) was prepared from oil, water, and Emultop partially
hydrolyzed soybean lecithin emulgator (2% w/w). R-Tocopherol (25
µM) was used as a reference antioxidant. The sample not containing
any antioxidant was used as a control. Concentrations of the samples
were 50 and 100 µg/g of oil based on the content of total phenolics.
Molar concentrations for 50 µg/g were 212-727 µM and for 100 µg/
g, 424-1454 µM. Samples were incubated at 37 °C for 6 days. The
inhibition against emulsion oxidation was calculated at day 6 by
measuring the formation of conjugated hydroperoxide dienes and
hexanal. The absorbance was measured at 234 nm by a Perkin-Elmer
λ 25 UV-vis spectrophotometer. For analysis of conjugated hydrop-
eroxide dienes a sample of emulsion (25 µL) or liposome (100 µL)
was dissolved in 5 mL of methanol. Measurements were done in
triplicates,andtheinhibitionofoxidationwasexpressedaspercentage(35,36).
For hexanal analysis a sample of emulsion (0.25 mL) or liposome (0.5
mL) was transferred into a headspace vial and the formation of hexanal
was measured according to the method by Frankel et al. (37). The
inhibition against formation of conjugated hydroperoxide dienes and
hexanal was calculated as
feruloyl-ꢀ-D-xylopyranoside, 4-nitrophenyl 4-O-feruloyl-ꢀ-D-xylopy-
ranoside, and 4-nitrophenyl 5-O-feruloyl-R-L-arabinofuranoside (Figure
3) were previously prepared according to the method of Mastihubova´
et al. (23) to be used for specific substrates for feruloyl esterases and
thus contained an additional 4-nitrophenyl moiety.
Analysis of Mixtures of Hydroxycinnamoyl Glucosides. For identi-
fication, NMR spectra were recorded with Varian Inova 500 and Varian
Mercury Plus 300 spectrometers (1H, 500 and 300 MHz; 13C, 125 and
75 MHz, respectively) in acetone-d6. The 2D NMR techniques (HSQC,
HSQC-TOCSY, and HMBC) were used for the identification of
products. The isomeric composition of the mixtures of acid glucosides
after the synthesis was determined with HPLC. The HPLC analysis
was performed using a Waters 717 plus autosampler, a Waters 600
pump, a Waters Symmetry C18, 5 µm, 4.6 × 150 mm column, and a
Waters 996UV spectrophotometric detector with detection at 280 nm.
Isocratic acetonitrile/water (18:82) was used as an eluent with flow
rate of 0.8 mL/min. MS and HRMS were detected with Bruker
microTOF using ESI.
inhibition (%) ) (A0 - A1)/A0 × 100
To verify that the feruloyl and sinapoyl glucosides contain only one
esterified hydroxycinnamoyl group per molecule, the glucoside ester
mixtures were hydrolyzed with Ultraflo L. The hydrolysis was
performed according to method by Vuorela et al. (31). The phenolic
sample (2 mL in methanol) was evaporated to dryness, and the enzyme
concentration was 0.1% (v/v) of the phenolic concentration in the final
volume (1100 µL in 0.02 M ammonium dihydrogen phosphate buffer,
pH 5.5). The hydrolyses were performed under nitrogen in a shaking
water bath at 37 °C. The HPLC analyses before and after the enzymatic
hydrolysis of hydroxycinnamic acid glucosides were performed ac-
cording to the method outlined by Koski et al. (32). The HPLC system
consisted of a 2690 separations module, a PDA 996 diode array detector,
and a Millennium32 software data module. The column was a Nova-
Pak C18 (150 × 3.9 mm, 4 µm, Waters, Millipore, Bedford, MA)
equipped with a C18 guard column. For detection, 320 nm (for
hydroxycinnamic acids) and 280 nm (generally for phenolics) were
the wavelengths recorded.
where A0 ) the absorbance for conjugated dienes and area for hexanal
of a control sample and A1 ) the absorbance for conjugated dienes or
area for hexanal of a tested sample.
Liposome Model System. The liposomes were prepared from soybean
lecithin (containing 40% phoshatidylcholine), and the concentration of
phosphatidylcholine in samples was 0.8 mg/mL. The inhibition against
liposome oxidation was calculated at day 3 by measuring the formation
of conjugated dienes and hexanal as described earlier. R-Tocopherol
(25 µM) was used as a reference antioxidant and a sample without any
antioxidant as a control. Concentrations of the samples were 4.2 and
8.4 µg/mL based on the content of total phenolics. Molar concentrations
for 4.2 µg/mL were 19-61 µM and for 8.4 µg/mL, 38-122 µM.
Oxidation was started by adding 3 µM cupric acetate. Samples were
stored in a shaking water bath (100 rpm, 37 °C) for 4 days. The
inhibition against oxidation was calculated at day 4 by measuring the
formation of conjugated hydroperoxide dienes and hexanal.
Total Phenolic Content. Samples (0.2 mL in methanol) were
evaporated to dryness. After that, 0.2 mL of deionized water, 1 mL of
Folin-Ciocalteu reagent (1:10), and 0.8 mL of disodium carbonate
solution (7.5%) were added. After 30 min, the total phenolic content
was measured at 765 nm by a Perkin-Elmer λ 25 UV-vis spectro-
Statistical Analysis. Statistical differences among antioxidant activity
were tested by multivariance analysis using Statgraphics Plus 3.0 (STCC
Inc., Rockville, MD). The significance level was p < 0.05. For the
statistical analysis samples were divided into groups and variances were
determined among the group. Sinapic acid and its glucoside esters

4800 J. Agric. Food Chem., Vol. 56, No. 12, 2008
Kylli et al.
Figure 4. HPLC analysis of the mixture of methyl feruloyl-D-glucopyranosides. The upper chromatogram is obtained before the enzymatic hydrolysis and
the lower one after the hydrolysis. Detection was at 320 nm. The main component in the mixture was methyl 6-O-feruloyl glucoside (62.4%).
Figure 5. HPLC analysis of the mixture of methyl sinapoyl-D-glucopyranosides. Methyl 3-O-sinapoyl-glucosides were tentatively identified due to lack of
pure isomer for peak identification. The upper chromatogram is obtained before enzymatic hydrolysis and the lower one after the hydrolysis. Detection
was at 320 nm. The main component in the mixture was methyl 6-O-sinapoyl glucoside (55.2%).
constituted one group. The mixture of feruloyl glucosides, 2-O-feruloyl-
glucoside, 3-O-feruloyl-glucoside, and 6-O-feruloyl-glucoside, consti-
tuted another group. A third group was formed from 5-hydroxyferulic
acid, ferulic acid dehydrodimer, ferulic acid, and the mixture of feruloyl
glucosides. 4-Nitrophenyl feruloyl glycosides were compared with each
other and with free ferulic acid.
and after the hydrolysis are equal, indicating that all glucoside
esters in the mixtures contained one hydroxycinnamoyl moiety
per molecule. The last peak in both chromatograms is tentatively
identified as 4-O-feruloyl- and 4-O-sinapoyl-6-acetyl glucoside.
In LC-MS, the observed m/z values measured with HRMS
(ESI/TOF) of Na+ adducts of all pure isomers of ester
conjugates between hydroxycinnamic acids and methyl glu-
copyranoside were identical: 393 and 423 for feruloyl and
sinapoyl conjugates, respectively. When referenced to pure
regioisomers synthesized, the main esterified positions in the
glucoside were 6-O and 2-O (Figures 4 and 5). The HRMS
and NMR results will be published and discussed elsewhere.
RESULTS
Characterization of Hydroxycinnamic Acid Glycoside
Esters. The HPLC analysis of mixtures of feruloyl- and
sinapoyl-glucoside esters revealed that they contained two main
and two minor compounds (Figure 4). The percentage composi-
tion of the mixtures is shown in Figures 4 and 5. The mixtures
were hydrolyzed with Ultraflo L, which is a multicomponent
cellulolytic and xylanolytic enzyme preparation, and the hy-
drolysates were analyzed by HPLC. The mixtures contained no
free phenolic acids before the hydrolysis. The corresponding
hydroxycinnamic acids were released during the hydrolysis, and
only minor amounts of feruloyl-glucoside esters remained,
whereas sinapoyl-glucoside esters were not hydrolyzed as
effectively. Quantification (data not shown) of the peak areas
from the chromatograms as ferulic acid or sinapic acid
equivalents revealed that the molar ratios of the acids before
The total phenolic content of the tested compounds was
determined by the Folin-Ciocalteu method (Table 1). Within
the group of similar structures, the total phenolic contents were
the same as calculated on the molecular weight basis. The
amount added of each compound to different antioxidant tests
was based on the measured total phenolic group content.
Radical Scavenging Activity. The radical scavenging activity
of the hydroxycinnamic acid glycoside esters was determined
at the concentration of 1 mg/mL (based on the total phenolic
content) (Figure 6). 6-O-Sinapoyl-glucoside had the best radical

Hydoxycinnamic Acid Glycoside Esters
J. Agric. Food Chem., Vol. 56, No. 12, 2008 4801
Table 1. Total Phenolics of Hydroxycinnamic Acids and Their Derivativesa
Ferulic acid and 2-O- and 6-O-feruloyl-glucosides were
significantly (p < 0.05) more effective antioxidants than 3-O-
feruloyl-glucoside. 2-O- and 6-O-feruloyl-glucosides inhibited
the conjugated hydroperoxide formation by over 85% and the
formation of hexanal by over 90%, whereas the antioxidant
effect of 3-O-feruloyl-glucoside was lower by 10-15%. Thus,
the same trend in antioxidant activity between different feruloyl
glucoside isomers, that is, 3-O-feruloyl-glucoside possessing
lower activity than the other two isomers, was detected both in
the radical scavenging test and in the liposome model system.
However, the antioxidative effect of feruloyl glucoside esters
in the liposome model system was not better than that of free
ferulic acid.
compound
total phenolics (mg/g ( SD)
sinapic acid
1043 ( 14
347 ( 58
407 ( 5
356 ( 13
988 ( 6
394 ( 2
407 ( 5
356 ( 13
367 ( 1
232 ( 8
225 ( 12
194 ( 6
241 ( 3
459 ( 2
308 ( 8
methyl sinapoyl glucoside mixture
methyl 2-O-sinapoyl-R-D-glucoside
methyl 6-O-sinapoyl-R-D-glucoside
ferulic acid
methyl feruloyl glucoside mixture
methyl 2-O-feruloyl-R-D-glucoside
methyl 3-O-feruloyl-R-D-glucoside
methyl 6-O-feruloyl-R-D-glucoside
4-nitrophenyl 2-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 4-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 5-O-feruloyl-R-L-arabinofuranoside
4-nitrophenyl ferulate
In the liposome model system, free ferulic acid was a more
potent antioxidant than 5-hydroxyferulic acid, the dehydrodimer
of ferulic acid, and the mixture of feruloyl glycosides. At the
concentration of 4.2 µg/mL, ferulic acid inhibited conjugate
diene formation by 87% and hexanal formation by 95%. The
dehydrodimer of ferulic acid and the mixture of feruloyl
glucosides had approximately the same antioxidant activities;
inhibition of the formation of conjugated dienes was 67-75%
and of hexanal was 84%. 5-Hydroxyferulic acid had the lowest
antioxidant activity toward conjugated diene (60%) and hexanal
formation (70%). At higher concentration the trend was the
same. The dehydrodimer of ferulic acid and the mixture of
feruloyl-glucosides were more active against lipid oxidation than
5-hydroxyferulic acid for both conjugated diene and hexanal
formation.
5-hydroxyferulic acid
ferulic acid dehydrodimer
a Ferulic acid and sinapic acid were used as standards for respective derivatives.
scavenging activity compared with other tested compounds. It
was statistically more active (96%) compared to free sinapic
acid (91%), the mixture of sinapoyl glucosides (91%), and 2-O-
sinapoyl-glucoside (91%).
The mixture of different feruloyl glucosides was more
effective in the radical scavenging test compared to the free
ferulic acid as well as pure isomers of glucoside esters (Figure
6). The mixture, which mainly contained 6-O- and 2-O-feruloyl-
glucosides, had the scavenging activity of 95%. Comparison
of the different pure isomers of feruloyl glucosides revealed
that the esterification to the primary C-6 hydroxyl group resulted
in significantly (p < 0.05) more effective radical scavenging
(86%) than esterification to the secondary C-2 positions (74%).
On the other hand, esterification to another secondary C-3
hydroxyl group resulted in much lower radical scavenging
activity (48%) than conjugation to C-2. The activity of free
ferulic acid was lower and of the same order of magnitude as
O-3-feruloyl-glucoside. The effect of conjugation on the radical
scavenging activity was also evaluated with 4-nitrophenyl 5-O-
feruloyl-R-L-arabinofuranoside, 4-nitrophenyl 4-O-feruloyl-ꢀ-
D-xylopyranoside, 4-nitrophenyl 2-O-feruloyl-ꢀ-D-xylopyrano-
side, and 4-nitrophenyl ferulate. The ferulic acid esterified to
the arabinofuranosyl moiety was the most effective, scavenging
activity being 61%. There was a slight difference compared to
feruloyl group linked to position C-4 (52%) and C-2 (48%) of
xylopyranoside or directly to 4-nitrophenol (49%). Control
experiments showed that radical scavenging activities of 1-m-
ethyl glucoside and 4-nitrophenyl glucoside were negligible
(2%). Overall, sinapic acid or ferulic acid esterified to the
primary hydroxyl of glycoside was a better radical scavenger
than esters to the secondary hydroxyl groups.
4-Nitrophenyl 5-O-feruloyl-arabinoside was the most power-
ful antioxidant among 4-nitrophenyl feruloyl glycosides at lower
concentration against both hexanal and conjugated diene forma-
tion. There was no significant difference between 4-nitrophenyl
glycosides of 2-O-feruloyl-xyloside, 4-O-feruloyl-xyloside, and
5-O-feruloyl-arabinoside at 8.4 µg/mL concentration.
Antioxidant Activity in Emulsion. The antioxidant activity
of hydroxycinnamoyl glycoside esters was examined in the
emulsion lipid oxidation model at 50 and 100 µg/g concentra-
tions (based on the total phenolic content) (Table 3). Generally,
the inhibition of hexanal formation was more efficient than the
inhibition of conjugated diene formation in the emulsion model,
similarly as in the liposome model. Comparison of the inhibition
of hexanal formation of sinapic acid and its glucoside esters
revealed that there are only minor differences between them.
The inhibition was over 85% for both tested concentrations.
Comparison of the inhibition of conjugate diene formation
showed the difference to be greater. The mixtures of sinapoyl
glucosides were the most effective against conjugated diene
formation. At both tested concentrations 6-O-sinapoyl-glucoside
was a more potent antioxidant than 2-O-sinapoyl-glucoside.
Thus, 2-O and 6-O-sinapoyl-glucosides behave differently in
the inhibition of formation of conjugated dienes in the liposome
model and in the emulsion model as no difference was observed
between these isomers in the liposome model. The increase of
concentration had a positive effect on the conjugate diene
formation, whereas the inhibition of hexanal formation was
already efficient at 50 µg/goil concentration.
Of the ferulic acid derivatives, 5-hydroxyferulic acid scav-
enged the radicals most effectively, about 95%, thus showing
as a good scavenging activity as the mixture of feruloyl
glucosides.
Antioxidant Activity in Liposome. The antioxidant activity
was measured in the liposome model system at 4.2 and 8.4 µg/
mL concentrations (based on the total phenolic content). The
inhibition of hexanal formation was excellent (>90%) for all
tested compounds except 3-O-glucoside, 5-hydroxyferulic acid,
and ferulic acid dehydrodimer at 8.4 µg/mL concentration
(Table 2). The inhibition of conjugated diene formation was
generally lower than the inhibition of hexanal formation. There
were no statistical differences between the mixture of sinapoyl
glucosides, 2-O-sinapoyl-glucoside, 6-O-sinapoyl-glucoside, and
sinapic acid, except for the inhibition of conjugate diene
formation with the mixture of sinapoyl glucosides.
At the concentration of 50 µg/g, 6-O-feruloyl-glucoside was
the most powerful antioxidant for both hexanal and conjugated
diene formation compared with 2-O- and 3-O-feruloyl-gluco-
sides. Also at a concentration of 100 µg/g 6-O-feruloyl-glucoside
as well as free ferulic acid was more effective than the other
two glucoside isomers for hexanal formation, but not for
conjugated diene formation. The mixtures of feruloyl glucosides

4802 J. Agric. Food Chem., Vol. 56, No. 12, 2008
Kylli et al.
Figure 6. Radical scavenging activity (DPPH test) of hydroxycinnamic acids, their glycoside esters, and other derivatives (1 mg/mL).
Table 2. Inhibition of Lipid Oxidation As Measured by Formation of Conjugated Dienes and Hexanal in Lecithin Liposome Oxidation Model System with 4.2
and 8.4 µg/mL Hydroxycinnamic Acids and Their Derivativesa
conjugated dienes (%)
4.2 µg/mL 8.4 µg/mL
hexanal (%)
compound
4.2 µg/mL
8.4 µg/mL
sinapic acid
89.2 ( 2.1
84.0 ( 2.9
90.8 ( 0.9
91.3 ( 0.5
87.0 ( 2.7
67.6 ( 4.9
84.1 ( 0.8
67.7 ( 7.3
87.0 ( 0.8
81.3 ( 4.3
85.7 ( 5.9
88.4 ( 4.7
76.3 ( 5.4
60.1 ( 5.1
75.2 ( 5.1
nd
96.4 ( 0.3
93.4 ( 1.7
93.8 ( 4.4
94.0 ( 2.7
95.4 ( 1.2
84.3 ( 3.0
80.6 ( 14.3
78.4 ( 4.1
89.5 ( 6.8
90.5 ( 2.4
91.0 ( 4.3
92.5 ( 0.7
87.6 ( 3.8
70.1 ( 3.4
83.1 ( 5.0
nd
methyl sinapoyl glucoside mixture
methyl 2-O-sinapoyl-R-D-glucoside
methyl 6-O-sinapoyl-R-D-glucoside
ferulic acid
methyl feruloyl glucoside mixture
methyl 2-O-feruloyl-R-D-glucoside
methyl 3-O-feruloyl-R-D-glucoside
methyl 6-O-feruloyl-R-D-glucoside
4-nitrophenyl 2-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 4-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 5-O-feruloyl-R-L-arabinoside
4-nitrophenyl ferulate
87.1 ( 2.1
90.5 ( 1.8
91.2 ( 1.8
nd
95.9 ( 0.4
92.8 ( 3.1
93.0 ( 2.4
nd
79.9 ( 3.1
86.5 ( 2.2
73.7 ( 6.0
88.5 ( 2.2
89.0 ( 3.0
89.7 ( 3.0
91.1 ( 3.5
80.1 ( 4.0
69.6 ( 6.3
82.4 ( 3.0
92.2 ( 1.9
90.8 ( 4.6
83.8 ( 1.7
93.1 ( 2.4
94.3 ( 0.9
93.7 ( 1.1
95.3 ( 1.2
90.7 ( 2.1
76.9 ( 3.8
88.6 ( 3.3
5-hydroxyferulic acid
ferulic acid dehydrodimer
a Concentrations based on the total phenolic content (percent inhibition, mean ( SD).
were also effective antioxidants for both hexanal (>90%) and
conjugated diene (>78%) formation.
There was no statistical difference between ferulic acid, ferulic
the most effective antioxidant was 4-nitrophenyl ferulate at 50
and 100 µg/g concentrations (79 and 87%).
acid dehydrodimer, and 5-hydroxyferulic acid in the inhibition
of lipid oxidation. Their inhibitions were approximately 60%.
4-Nitrophenyl ferulate, 4-nitrophenyl 2-O-feruloyl-xyloside, and
4-nitrophenyl 5-O-feruloyl-arabinoside had no significant dif-
ference at 50 µg/g concentration. Their inhibition against hexanal
formation was 85-93%. At higher concentration 4-nitrophenyl
ferulate, 4-nitrophenyl 4-O-feruloyl-xyloside, and 4-nitrophenyl
5-O-feruloyl-arabinoside inhibited the hexanal formation at over
90%. 4-Nitrophenyl 2-O-feruloyl-xyloside had the lowest an-
tioxidant activity (79%). Against conjugated diene formation,
DISCUSSION
Radical Scavenging and Antioxidant Activity of Feruloyl
and Sinapoyl Glucosides. Glucoside esters were very potent
radical scavengers (DPPH test), with the inhibition over 85%
except for 2-O-feruloyl-glucoside and 3-O-feruloyl-glucoside.
In the DPPH test only the amount of hydroxyl groups is
significant in assessing radical scavenging activity. Therefore,
no difference between the feruloyl glucosides was anticipated,
contrary to the findings. A possible explanation for the lower

Hydoxycinnamic Acid Glycoside Esters
J. Agric. Food Chem., Vol. 56, No. 12, 2008 4803
Table 3. Inhibition of Lipid Oxidation As Measured by Formation of Conjugated Dienes and Hexanal in Emulsion Oxidation Model System with 50 and 100
µg/g Hydroxycinnamic Acids and Their Derivativesa
conjugated dienes (%)
50 µg/g 100 µg/g
hexanal (%)
compound
50 µg/g
100 µg/g
nd
99.8 ( 0.4
95.1 ( 5.2
94.4 ( 7.3
nd
95.4 ( 7.1
95.2 ( 6.2
81.5 ( 16.0
92.4 ( 10.9
79.0 ( 10.4
91.5 ( 2.6
95.2 ( 5.0
95.8 ( 1.7
94.7 ( 4.8
92.7 ( 3.4
sinapic acid
66.3 ( 4.2
90.3 ( 2.8
57.6 ( 8.1
83.4 ( 8.5
59.5 ( 4.2
78.3 ( 6.5
50.8 ( 6.7
72.6 ( 5.2
73.4 ( 11.9
55.3 ( 7.1
49.7 ( 8.1
64.4 ( 4.3
79.2 ( 5.3
71.6 ( 10.5
53.8 ( 19.6
nd
83.5 ( 6.9
94.6 ( 3.0
96.8 ( 1.4
92.0 ( 5.5
87.0 ( 7.1
91.7 ( 7.6
70.4 ( 5.4
68.4 ( 5.2
89.5 ( 8.0
85.9 ( 3.7
37.2 ( 8.0
93.2 ( 7.9
88.9 ( 7.8
93.1 ( 0.7
85.8 ( 4.5
methyl sinapoyl glucoside mixture
methyl 2-O-sinapoyl-R-D-glucoside
methyl 6-O-sinapoyl-R-D-glucoside
ferulic acid
methyl feruloyl glucoside mixture
methyl 2-O-feruloyl-R-D-glucoside
methyl 3-O-feruloyl-R-D-glucoside
methyl 6-O-feruloyl-R-D-glucoside
4-nitrophenyl 2-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 4-O-feruloyl-ꢀ-D-xyloside
4-nitrophenyl 5-O-feruloyl-R-L-arabinoside
4-nitrophenyl ferulate
90.1 ( 3.8
54.7 ( 9.0
76.0 ( 15.0
nd
79.0 ( 10.0
64.6 ( 10.1
78.0 ( 11.1
59.1 ( 7.7
76.4 ( 6.5
76.9 ( 10.9
71.9 ( 5.4
86.9 ( 4.2
67.7 ( 13.2
66.2 ( 13.5
5-hydroxyferulic acid
ferulic acid dehydrodimer
a Dosages based on the total phenolic content (percent inhibition, mean ( SD).
radical scavenging activity toward DPPH radical when using
2-O- and 3-O-feruloyl glucosides might be a slight difference
in the solubility in the test solvent, methanol. The presence of
the -CHdCHsCOOH chain in hydroxycinnamic acids ensures
hydrogen-donating ability and subsequent radical stabilization
(38). Ortho substitution with electron-donating alkyl or methoxy
groups increases the stability of the radical formed from the
phenolic compound and its antioxidant potential (39). There was
no difference between sinapic acid and sinapic acid conjugates.
In the emulsion and liposome oxidation, ferulic acid and its
derivatives had similar antioxidant activities, indicating that the
introduction of a sugar moiety to the hydroxycinnamic acid does
not reduce the capability for the antiradical potential. Moreover,
the present results as well as our previous studies on hydroxy-
cinnamic acids (40) suggest that the naturally existing glycoside
esters are at least as potent as the free phenolic acids. More
hydrophilic glucoside esters of hydroxycinnamic acids are more
active radical scavengers compared with free forms of acids.
These findings contradict earlier reports. Tominaga et al. (41)
found that the introduction of a ꢀ-glucose moiety to caffeic and
ferulic acids decreased their radical scavenging activities due
to increased hydrophilicity and permeability in aqueous solutions
in the DPPH test. According to Cos et al. (42) caffeic acid had
the highest scavenging activity followed by ferulic and sinapic
acids, whereas the esterification of caffeic acid with quinic acid
decreased the activity.
Feruloyl-ꢀ-D-glucopyranosides, sinapoyl-ꢀ-D-glucopyrano-
sides, and caffeoyl-ꢀ-D-glucopyranosides were reported to be
weaker antioxidants than free corresponding phenolic acids
toward the inhibition of oxidation of ꢀ-carotene-linoleic acid
emulsions (41, 43). In our studies, the hydroxycinnamic acid
esters were as potent antioxidants as the free acids also toward
the oxidation of liposomes and emulsions. In general, 6-O-
feruloyl-glucoside exhibited the highest antioxidant activity
followed by ferulic acid, 2-O-feruloyl-glucoside, and 3-O-
feruloyl-glucoside, thus complying with the original hypothesis.
With sinapic acid conjugates no significant difference was seen.
This may be explained by the significant antioxidant potency
of sinapic acid and its derivatives compared to other hydroxy-
cinnamates. Sinapic acid and its derivatives behave similarly
in liposomes and emulsions. The mixture of sinapoyl glucosides
was the most efficient at emulsions, whereas in liposomes they
were less potent. Sinapic acid has been also earlier shown to
be a more effective antioxidant than ferulic acid, which in turn
is more effective than p-coumaric acid (42). Nystro¨m et al. (3)
have demonstrated that in bulk and emulsified methyl linoleate
steryl ferulates had a similar activity against lipid oxidation as
ferulic acid. Feruloylated arabinoxylotrisaccharides from wheat
enhanced the activity toward LDL oxidation, whereas the
activity was reduced compared to ferulic acid in the DPPH
test (44, 45). Antioxidants trapped near the surface of the
liposomal membrane are better antioxidants as shown with hexyl
and octyl ferulates being more effective than shorter alkyl
esters (41, 43). In emulsions the lipophilic antioxidants of low
hydrophilic-lipophilic balance are favored (37). The lipophilic
antioxidants are adequately surface active to be located at the
oil-water interface, where they are able to inhibit lipid
oxidation, whereas hydrophilic antioxidants are located in the
aqueous phase and are consequently less effective against lipid
oxidation (46). The oil-water interface appears to be significant
for antioxidants to function as chain-breakers. Emulsifiers, which
are amphiphilic, are located at the oil-water interfaces in
emulsions and can solubilize antioxidants, thus enabling polar
antioxidants to get in contact with lipids. Sto¨ckmann et al. (47)
showed that emulsions containing partially hydrolyzed soybean
lecithin as emulsifier solubilized gallic acid more efficiently than
its more hydrophobic derivative ethyl gallate. Feruloyl and
sinapoyl glucosides are more hydrophilic than their free forms.
Due to the ability of soybean lecithin to solubilize hydrophilic
compounds, tested glucosides were effective antioxidants.
Altering the pH changes the solubility of hydroxycinnamic acids
to the aqueous phase and lowering the pH might be related to
the dissociation of the functional group. Schwarz et al. (48)
observed that the concentrations of ferulic acid and caffeic acid
decreased in the aqueous phase of water/oil systems when the
pH was lowered from 7.0 to 3.0. Introduction of sugar to the
phenolic compounds would make them more hydrophilic and
inhibit them from reaching the oil-water interface (49). These
findings were confirmed in the present study, where the tested
hydrophilic antioxidants were not as effective in emulsions as
in liposomes. The pH in the present study was 5.0 in the
liposomes and neutral in the emulsion.
Antioxidant Activity of 4-Nitrophenyl Feruloyl-Glycosides.
To further test the hypothesis of conjugation of glycosides to
hydroxycinnamic acids resulting in similar antioxidant activity
as free acids, different 4-nitrophenyl feruloyl-glycosides were
investigated. 4-Nitrophenyl 5-O-feruloyl-arabinoside was the
most powerful antioxidants among the 4-nitrophenyl glycosides.
4-Nitrophenyl 4-O-feruloyl-xyloside was the second most
effective, whereas 4-nitrophenyl 2-O-feruloyl-xyloside was the

4804 J. Agric. Food Chem., Vol. 56, No. 12, 2008
Kylli et al.
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weakest antioxidant in general. 4-Nitrophenyl ferulate had an
activity similar to that of free ferulic acid. The 4-nitrophenyl
moiety did not change the antioxidant activity of ferulic acid,
although it changes the hydrophilicity. In liposomes at a
concentration of 100 µg/g all 4-nitrophenyl derivatives had equal
antioxidant activity. At lower concentration, the order of the
activity was 4-nitrophenyl 5-O-feruloyl-arabinoside > 4-nitro-
phenyl 4-O-feruloyl-xyloside > 4-nitrophenyl 2-O-feruloyl-
xyloside > 4-nitrophenyl ferulate. In emulsions 4-nitrophenyl
5-O-feruloyl-arabinoside and 4-nitrophenyl ferulate inhibited the
most hexanal and conjugated diene formation. In all tests, there
was no difference between different 4-nitrophenyl
derivatives.
Antioxidant Activity of Other Ferulic Acid Derivatives.
5-Hydroxyferulic acid and ferulic acid dehydrodimer have
different solubilities in the lipid phase and thus exhibit different
orientations in liposome and emulsions. Paiva-Martins et al. (50)
discovered that the oxidative attack seems to occur from the
aqueous phase initiating lipid peroxidation. The antioxidant
activity of phenolic compounds in liposomes depends both on
the location and on the orientation of the antioxidant in the
system. Lipophilic antioxidants can act both by scavenging of
aqueous peroxyl radicals and by scavenging of lipid peroxyl
radicals within the liposomal membrane. The more hydrophilic
5-hydroxyferulic acid has a lower hydrophilic-lipophilic bal-
ance, and therefore it was a more powerful antioxidant compared
with ferulic acid and ferulic acid dehydrodimer. In liposomes,
the order was reversed, with ferulic acid dehydrodimer being
the most effective. In acidic conditions, phenolic acids are weak
antioxidants, whereas with increasing pH their antioxidant
activity increases (17). The explanation for the better antioxidant
activity in basic conditions is due to a rapid electron transfer
from anionic phenolic acids to peroxyl radicals. Hydrogen
abstraction and electron transfer in the antioxidant reactions
depend on the conditions, such as pH value and the stability of
the intermediate radicals (49).
In conclusion, sinapic acid and its derivatives were the most
effective antioxidants. The esterification to glycosides did not
decrease the antioxidant activity of ferulic and sinapic acids.
In most cases, the mixture of ferulic acid glucosides or ferulic
acid esterified to the position C-6 of methyl glucopyranoside
was more potent lipid oxidation inhibitor compared with esters
on positions C-2 and C-3. 4-Nitrophenyl 5-O-feruloyl-arabino-
furanoside was also the most effective among the 4-nitrophenyl
glycosides. The most likely explanation is that hydroxycinnamic
acids esterified to the primary hydroxyls in glucopyranoside and
arabinofuranoside are able to move more freely than in other
isomers, thus enabling them to function as antioxidants more
efficiently. Our studies indicate that naturally existing or
synthetic glycoside esters of phenolic acids are potent antioxi-
dants and that the antioxidant activity also depends on the
conjugation.
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ACKNOWLEDGMENT
We thank Klaus Koivu for preparing the feruloyl- and
sinapoyl-glucosides, Dr. Ma´ria Mastihubova´ for a sample of
4-nitrophenyl ferulate, and Minnamari Edelmann for skillful
assistance in antioxidant activity testing.
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Received for review January 31, 2008. Revised manuscript received
March 5, 2008. Accepted March 18, 2008. Support from the University
of Helsinki Research Funds is gratefully acknowledged.
JF800317V