Article
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6939
Table 2. Deacetylation of 4-O-Acetyl-Protected Hydroxycinnamic Acid Xylan
Esters by NaBH4 in DMA/LiCl
as a catalyst under microwave heating; however, the reaction did
not succeed. As it is well-known that cellulose and hemicelluloses
can be dissolved in DMA/LiCl solution system (18, 23), we
changed the approach in which xylans were first dissolved in
DMA/LiCl (4% w/w) solution, followed by the addition of
pyridine, DMAP, and acetyl-protected acid chlorides at room
temperature under argon and finally stirring the reaction mixture
at 60 °C overnight for the esterification (Figure 1, Table 1). In the
optimized conditions shown in Table 1, the quantities of pyridine
and DMAP were 1.5 mol equiv to acid chlorides and 5 mol % of
the xylans, respectively. The amounts of 4-O-acetylferulic acid
chlorides and 4-O-acetylsinapic acid chlorides in the esterification
of oat spelt arabinoxylan were 0.5, 1.0, 1.5, 3.0, 1.0, and 1.5 mol
equiv to XU (anhydroxylose units, i.e., the amount of hydroxyl
functionality in xylan, Table 1), respectively. In the case of birch
wood glucuronoxylan, the derivatization was carried out with 1.0
and 1.5 mol equiv of 4-O-acetylferulic acid chlorides to XU.
Weight Percent Gain and Solubility of Ester Adducts. Since
some loss of weight of xylans occurred during the esterification
step, we employed weight percent gain (20) to describe the
amount of substitution in the polymers instead of calculating
yields to quantitatively follow the modification ofxylans. The loss
of weight is obviously due to the salts and minor sugar compo-
nents present in commercially available oat spelt and birch wood
xylans.
substrate (no.)
temp (°C)
time (h)
WPG (%)a
DSb
compd (no.)c
AcFA-AX (1)
AcFA-AX (2)
AcFA-AX (3)
AcFA-AX (4)
AcSA-AX (5)
AcSA-AX (6)
AcFA-GX (7)
AcFA-GX (8)
25
25
25
25
40
40
40
40
72
72
-13
-18
-24
-27
-9
-21
-10
-19
0.05
0.36
0.45
0.89
0.09
0.26
0.05
0.39
FA-AX (9)
FA-AX (10)
FA-AX (11)
FA-AX (12)
SA-AX (13)
SA-AX (14)
FA-GX (15)
FA-GX (16)
72
72
72
72
100
100
a WPG = weight percent gain (see Materials and Methods). b Determined with
HPLC after alkali hydrolysis (1% NaOH at 25 °C in 12 h). c FA-AX = ferulic acid
oat spelt arabinoxylan ester; SA-AX = sinapic acid oat spelt arabinoxylan ester;
FA-GX = ferulic acid birch wood glucuronoxylan ester.
quantity of hydroxycinnamic acid released in basic hydrolysis of the
hydroxycinnamic acid xylan esters (Figure 1, Table 2) using HPLC.
Accordingly, the polymers (70 mg) were hydrolyzed in 3 mL of 1% NaOH
at 25 °C in 12 h, followed by acidification to pH 2-3 by 1 M HCl and
extraction with EtOAc. After workup the liberated ferulic and sinapic acid
contents were determined by the HPLC system consisting of a Waters 2690
separation module, a PDA 996 diode array detector, and Empower 2
(build 2154) software. The column used was a 150 ꢀ 3.9 mm, 4 μm, Nova-
Pak C18 with a C18 guard column (Waters, Millipore, Bedford, MA).
Samples were dissolved in 50 mM ammonium dihydrogen phosphate, pH
2.6. For detection, the wavelength at 320 nm was used. The HPLC analysis
The WPG values presented in Table 1 clearly show that an
increase in the molar ratio of acid chlorides to anhydroxylose
units in the esterification leads to an increase in the WPG values.
The rising trend in WPG values is apparently linked to the
increase in the degree of substitution to the xylan backbone (or
to the Araf or meGlcpA side chains). The only exception was
found in the case of compound 4, which gave a lower WPG value
than expected. This could be due to its higher solubility in the
solvents used for the precipitation.
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was performed according to the method outlined by Kahkonen et al (21).
Commercial ferulic and sinapic acids were used as standards. DS was also
estimated by means of 1H NMR spectroscopy by comparing the signals
between 1-H xylan and R-H and β-H of the hydroxycinnamic acid moiety.
The data were in accordance with the DS determined with HPLC by
hydrolysis except in the case of compounds 4 and 12 (with DS of 0.89,
Tables 1 and 2) since in these samples the peaks were too broad for
accurate determination.
Molar Mass Analysis. The ferulic acid ester with DS of 0.36 (com-
pound 10, Table 1) was analyzed with high-performance size exclusion
chromatography (HPSEC) using DMSO with 0.01 M LiBr as eluent (22).
The HPSEC system consisted of an integrated autosampler and pump
module (GPCmax, Viscotec Corp., Houston, TX), two linear columns,
8 ꢀ 300 mm, Shodex LF-804, and a guard column, 4.6 ꢀ 10 mm, Shodex
LF-G (Showa Denko, Tokyo, Japan), a UV detector at λ0 = 280 nm
(Waters 486 tunable absorbance detector, Milford, MA), a combined light
scattering and viscometric detector (270 dual detector, Viscotek Corp.),
and a refractive index (RI) detector (VE 3580, Viscotek Corp.). The flow
rate was 1 mL/min and injection volume 100 μL. The samples (4 mg/mL)
were dissolved in eluent at room temperature for 4 days to ensure sufficient
solubility. All of the samples were filtered before analysis with 0.45 μm
syringe filters (GHP Acrodisc 13, Pall Corp., Ann Arbor, MI). OmniSEC
4.5 was used for data processing.
Antioxidative Activity. Antioxidant activities of xylan esters were
tested by using the emulsion lipid oxidation test according to Kylli et al. (11).
Emulsions were prepared from oil (10% o/o), water, and soybean lecithin
emulgator (2% w/w). Tested samples were suspended to the flasks at a
concentration of 500 μg of xylan esters/g of oil. Samples were incubated at
37 °C for 6 days. The inhibition against emulsion oxidation was calculated
at day 6 by measuring the formation of hexanal (primary lipid oxidation
product) by headspace gas chromatography and conjugated hydroperoxide
dienes (secondary lipid oxidation product) by measuring the absorbance
spectrometrically at 234 nm as described earlier (11). Analyses were made in
triplicate, and R-tocopherol was used as a reference.
In determination of the solubility properties of the polymers, it
was found that all of the prepared oat spelt arabinoxylan esters
(compounds 1-6, Figure 1) were soluble in DMA/LiCl while the
birch wood glucuronoxylan esters (compounds 7 and 8), surpris-
ingly, were only partially soluble in that solvent even if stirred at
130 °C for 6 h. In addition, all of the compounds were insoluble in
common polar solvents (chloroform, ethanol, tetrahydrofuran,
toluene, ethyl acetate, or water) and nonpolar solvents (hexane)
except compound 4, which was slightly soluble in THF and
chloroform (10-15%). In the preparation of the NMR samples
we also found that the solubility of the samples in DMSO
increased with the amount of acid chlorides applied in the
esterification. Thus, compounds 1, 2, 3, 5, 6, 7, and 8 were only
partially soluble in DMSO at room temperature, while the ester
compounds 2, 3, and 4 were completely soluble when heated up to
100 °C (NMR tube, 50 mg of compounds in 0.7 mL of DMSO-d6).
Compound 4 was completely soluble even at room temperature,
which could further explain its low WPG value obtained.
Deacetylation of Xylan Derivatives. In our previous studies we
found that pyrrolidine removes the acetyl group selectively from
the 4-O-acetyl-protected feruloyl- and sinapoyl glucoside esters in
homogeneous conditions (11). However, in the case of xylan
esters, no reaction took place. When using 1% sodium hydroxide
(with compound 3, Figure 1), a complete deprotection took place
in 45 min at 0 °C, but unfortunately, a significant amount of
ferulic acid was also liberated. For example, the DS of compound
10 with DS of 0.36 (Table 2) was reduced to less than 0.05
according to 1H NMR.
RESULTS AND DISCUSSION
Esterification of Oat Spelt Arabinoxylan (AX) and Birchwood
Glucuronoxylan (GX). To prepare moderately esterified xylans
with hydroxycinnamic acids (ferulic acid and sinapic acid), the
route described in Figure 1 for their synthesis was developed.
In our first experimental setup, we studied the reaction in
heterogeneous conditions using pyridine as a solvent and DMAP
The selective deesterification of the xylan esters was ultimately
achieved by using NaBH4 in DMA/LiCl in large excess (over
10 mol equiv) (Table 2). Thus, for the deacetylation, xylan esters