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,3
Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, School of Medicine,
Department of Viticulture and Enology, and
Department of Nutrition, University of California, Davis, CA; and
Sequoia Sciences, San Diego, CA
**
*
3To whom correspondence should be addressed. E-mail: fmsteinberg{at}ucdavis.edu.
ABSTRACT
Recent epidemiologic studies have associated nut consumption with a
reduced incidence of cardiovascular mortality. However, little is known
about the contribution of nut polyphenols to antioxidant and
cardiovascular protection. In this investigation, polyphenol-rich
extracts from English walnuts (Juglans regia) were
studied and compared with ellagic acid for their ability to inhibit in
vitro plasma and LDL oxidation, as well as their effects on LDL
-tocopherol during oxidative stress. In addition, the Trolox
equivalent antioxidant activity (TEAC) was determined and liquid
chromatography electrospray detection mass spectrometry (LC-ELSD/MS)
analyses of the walnut extracts were performed. 2,2'-Azobis'(2-amidino
propane) hydrochloride (AAPH)-induced LDL oxidation was significantly
inhibited by 87 and 38% with the highest concentration (1.0
µmol/L) of ellagic acid and walnut extract,
respectively. In addition, copper-mediated LDL oxidation was
inhibited by 14 and 84% in the presence of ellagic acid and walnut
extract, respectively, with a modest, significant LDL -tocopherol
sparing effect observed. Plasma thiobarbituric acid reacting substance
(TBARS) formation was significantly inhibited by walnut extracts and
ellagic acid in a dose-dependent manner, and the extracts exhibited
a TEAC value greater than that of -tocopherol. LC-ELSD/MS analysis
of the walnut extracts identified ellagic acid monomers, polymeric
ellagitannins and other phenolics, principally nonflavonoid compounds.
These results demonstrate that walnut polyphenolics are effective
inhibitors of in vitro plasma and LDL oxidation. The polyphenolic
content of walnuts should be considered when evaluating their
antiatherogenic potential.
KEY WORDS: • polyphenol • ellagic acid • LDL • antioxidant • English walnuts
Recent epidemiologic studies have shown that consuming diets rich in
plant-derived foods that are high in phenolic compounds, even while
consuming high amounts of saturated fatty acids (1
), is
associated with a reduced incidence of cardiovascular mortality
(2
–5
). Polyphenolics have been shown to possess free
radical–scavenging and metal-chelating activity in addition to
their reported anticarcinogenic properties (6
). These
plant-based, nonnutrient phytochemicals may have a protective
effect on the susceptibility of LDL to oxidative modification and
ultimately, on atherosclerosis.
The oxidative modification and inflammation hypothesis of atherogenesis
is widely accepted and supported by experimental data in
hypercholesterolemic animal models and human epidemiologic studies,
recently reviewed by Chisolm and Steinberg (7
).
Atherogenesis is a multifactorial process that includes oxidatively
modified LDL, which triggers pathological events through multiple
pathways, leading to atherosclerosis (8
). Numerous in
vitro studies using a variety of oxidation methods and measurements
have shown that polyphenolics from red wine (9
), green tea
(10
) and chocolate (11
) can inhibit LDL
oxidation. In vivo, it is unclear whether diet-derived
polyphenolics can indeed influence the atherogenic process, but it is
thought that the antioxidant potential of plant-derived foods may
be one factor in reducing cardiovascular risk (12
).
A correlation between nut consumption and a reduced incidence of
ischemic heart disease has been observed, most notably by the Adventist
Health Study, the Iowa Women’s Health Study and the Nurses’ Health
Study (13
–15
). Consumption of walnuts has favorable
effects on human serum lipid profiles, with a decrease in total and LDL
cholesterol as well as triglycerides (16
–19
) and an
increase in HDL cholesterol and apolipoprotein A1 (20
).
Tree nuts are an important source of beneficial dietary lipids, and a
potentially rich source of phenolic compounds that contribute to
antioxidant capacity. However, there has been very little investigation
into the contribution of nut polyphenols to antioxidant protection and
reduction in cardiovascular risk. This study, therefore, sought to
determine the antioxidant capacity of walnut polyphenolic compounds in
vitro. Walnut polyphenolics are found in the highest concentration in
the pellicle, the thin tan-brown "skin" that lines the meat of
the nut. Thus, this paper reports on a brief characterization of the
polyphenolic-rich extract of English walnuts, its relative
antioxidant activity, and demonstrates the ability of this extract to
significantly inhibit plasma and LDL oxidation.
MATERIALS AND METHODS
Materials.
1,1,3,3-Tetramethoxypropane [malondialdehyde
(MDA)4
], 2,2'-azobis'(2-amidino propane) hydrochloride (AAPH), HPLC grade
ethyl alcohol and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox, Hoffman La-Roche) were purchased from Aldrich (Milwaukee,
WI). Unless otherwise noted, all other chemicals and reagents were
purchased from Sigma Chemical (St. Louis, MO). The English walnuts (a
mixture of Hartley and Chandler cultivars) were purchased from the
Mariani company (Winters, CA). For the oxidation experiments, ellagic
acid and gallic acid standards were dissolved in a 10% ethanol
solution and subsequent dilutions made in deionized H2O.
Catechin was dissolved in water, adjusted with 11.6 mol/L HCl (Fisher,
Pittsburg, PA) to a pH of 3.5, and vortexed for at least 10 min. The
catechin was used as a positive control due to its previously described
ability to inhibit LDL oxidation (21
, 22
). For the
antioxidant activity assay, the compounds and extracts were diluted in
100% HPLC grade ethyl alcohol.
Preparation of walnut polyphenolic extracts and Folin assay.
The walnuts were frozen for 24 h; the shelled kernel was then
immersed in a solution of 75% acetone + 526 µmol/L
sodium metabisulfite and the headspace purged with N2
(nitrogen) to guard against oxidation. After incubation at 4°C for
24 h, solutions were decanted, resulting in a cold extract. To
obtain a heat extract, an additional step of gentle refluxing of the
decanted solution under N2 gas for 24 h was added. To
remove all lipids, the extraction solutions were evaporated under
reduced pressure to remove all acetone, and methanol (50% aqueous,
v/v) was added; the solutions were then extracted three times with
hexanes. To prevent oxidation, the extract solutions were lyophilized
to a dry powder and stored at -20°C. Before use, the lyophilized
powder was dissolved into a 10% dimethyl sulfoxide solution and
ultrapure water. The concentration of the dissolved extracts was
determined using the Folin-Ciocalteu assay, a colorimetric assay
for total phenolics (23
). Briefly, the Folin-Ciocalteu
reagent was mixed with serial dilutions of walnut extract and gallic
acid standards. After a short incubation, 1.9 mol/L sodium carbonate
was added followed by a 1-h incubation. The absorbance at 765 nm was
obtained and compared with that from gallic acid standards, with
results expressed in gallic acid equivalents (GAE).
Antioxidant assay.
The antioxidant activity of the test compounds and extracts was
determined using a 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS) cation decolorization assay as previously described
(24
). Briefly, Trolox, a water-soluble vitamin E
analog, was used as a standard and thus the results are expressed as
Trolox equivalent activity capacity (TEAC). The ABTS radical cation
solution was produced by the addition of potassium persulfate to a
final concentration of 2.45mmol/L in 7mmol/L ABTS and incubated for
12–16 h, in which time the radical cation stabilizes. Absorbance at
734nm was obtained 4 min after the addition of the compounds and
extracts. This assay was carried out in triplicate on three separate
days.
LC-ELSD-MS of walnut extracts.
A HPLC method coupled to electrospray ionization mass spectrometry (LC-ELSD/MS) was used for characterization of the walnut phenolics. A Waters (Milford, MA) 600 binary pump fed a Gilson (Middleton, WI) 215 multiple injection autosampler with an injection volume of 20 µL onto a Betabasic C18 column (4.6 x 50 mm, 5-µm particle size) from Western Analytical (Temecula, CA). The mobile phase was a water (A)/acetonitrile (B) gradient both containing 0.1% acetic acid, starting with 5% B and progressing via linear gradients to these amounts at these times, 1 min 5%; 8.5 min 75%; 9 min 95%. After 1 min at 95% B, the solvent was equilibrated back to 5% B. The flow was 1.2 mL/min, and of this, 0.1 mL/min was delivered to the mass detector. The MS data were acquired on a LCT orthogonal TOF spectrometer (Micromass; Beverly, MA) fitted with an eight-way multiplexed electrospray interface (MUX). In these analyses, each liquid stream was sampled for 0.1 s with mass spectra acquired from 200-1000 Da into eight simultaneously open data files synchronized with the spray being sampled. The time taken to move to the adjacent sampling position was 0.05 s. This cycle produced a data point for each spray every 0.15 s. The LCT and MUX were operated under MassLynx V3.4. The ionization conditions and energy levels for both electrospray negative and positive modes were as follows: desolvation temperature, 380°C; source temperature, 120°C; ion energy, 35 eV; extraction cone, 2 eV; desolvation gas flow, 1200 L/h; and cone gas flow, 11 L/h. Additionally, the energy conditions that differed for electrospray negative and positive modes were capillary 3000 and 3500eV, sample cone 45 and 50eV, and RF lens 500 and 350ev, respectively. The instrument was calibrated in both ionization modes over a 150- to 1000-Da mass range using a 0.02 g/L solution of poly-DL-alanine dissolved in methanol.
Plasma and LDL isolation.
Blood from five healthy normocholesterolemic adult volunteers was
collected into vacutainer tubes containing EDTA and centrifuged (833
x g for 10 min) to isolate plasma. The plasma was
pooled and stored at -70°C in 2-mL aliquots and used for all
subsequent experiments. LDL was isolated by density centrifugation in a
Sorvall RC-120GX Micro-Ultracentrifuge using a Sorvall (Newtown,
CT) S120-AT2 rotor, based on the method of Brousseau et al.
(25
). The LDL layer was removed and dialyzed against 4 L
of PBS, pH 7.4, and purged with nitrogen for 12–18 h. The PBS was
treated with 10–20 g/L of Chelex (Bio-Rad, Hercules, CA), which was
removed before use in dialysis. After dialysis, the cholesterol
concentration in the pooled LDL sample was determined using a
Cholesterol/HP assay kit (Roche Molecular Biochemicals, Indianapolis,
IN) as described by the manufacturer. The LDL fraction was used
immediately or purged with nitrogen and stored at 4°C for no >48 h.
Copper-mediated LDL oxidation.
To evaluate the ability of the test compounds and extracts to inhibit
copper (Cu)-mediated LDL oxidation, conjugated dienes (CD) were
continually monitored (5-min intervals) at 37°C by obtaining UV
absorption at 234 nm (26
). LDL oxidation was initiated by
adding copper sulfate at a final concentration of 5
µmol/L to 75 µg of LDL cholesterol in
a volume of 1 mL. The purified test compounds (ellagic acid and
catechin obtained from Sigma Chemical) and walnut extracts were added
immediately before the addition of the oxidant, using the extracts at a
final concentration of 0.1–1.0 µmol/L GAE and the
purified compounds at a final concentration of 0.5–1.0
µmol/L. Concentrations of 0.1 and 0.5
µmol/L represent physiologic plasma levels of phenolic
compounds achievable after ingestion of phenolic-rich foods, and
1.0 µmol/L was chosen to determine whether a
dose-response effect might be seen with higher levels of the
phenolic compounds. All LDL experiments were performed twice in
triplicate. The lag time in the presence or absence of the test
compounds was determined to be the intercept of the slopes for the lag
and propagation phases, and was compared with the control oxidized LDL
to determine the percentage of LDL oxidation inhibition.
HPLC determination of -tocopherol.
The LDL was oxidized in the same manner as described above with
extracts and ellagic acid, final concentration of 0.5 and 1.0
µmol/L, respectively, added immediately before or
exactly 10 min after the addition of Cu. This mixture was incubated in
a 37°C water bath, aliquots removed at various time points and
oxidation stopped with the addition of BHT. The LDL -tocopherol was
extracted with hexane, the organic phase removed and evaporated under a
low pressure nitrogen stream. The resulting sample was reconstituted in
200 µL of HPLC mobile phase (50:50
acetonitrile/ethanol) and transferred to amber injection vials for
direct injection and separation using a C-18 Ultramex 150 x 4.6
mm, 5-µm particle size reverse phase column
(Phenomenex, Torrence CA), with -tocopherol detected at 293 nm
wavelength as previously described (27
).
Azo-initiated LDL and plasma oxidation.
LDL oxidation (75 µg of LDL cholesterol in a 1 mL
volume) was initiated by the addition of AAPH, a free radical
generator, at a final concentration of 1 mmol/L and CD formation
measured as described above. The test compounds and walnut extracts
were used at the same concentrations as described for the
Cu-mediated experiments. In another experiment, plasma oxidation
products were measured by means of thiobarbituric acid reacting
substance (TBARS) formation as previously described and carried out
with slight modifications (28
). Plasma (100
µL) oxidation was initiated with the addition of AAPH
at a final concentration of 50 mmol/L in the presence of the walnut
extracts or ellagic acid. Samples were incubated for 4 h at 37°C
and the oxidation subsequently stopped with the addition of BHT. TBARS
concentration was determined by UV absorption at 532 nm, compared with
a MDA standard curve, and results expressed as micromolar MDA
equivalents.
Statistical analysis.
All values are expressed as means ± SD. A parametric one-way ANOVA was performed on oxidation data using Systat 9 (Chicago, IL). To determine significant difference from control values, a Tukey multiple comparison post-hoc test was performed. Differences with P < 0.05 were considered significant.
RESULTS
Total phenols in 50 g of walnuts (
8–9 shelled walnuts) as
determined by the Folin-Ciocalteu assay, contained 802 mg GAE. Mass
spectrometry (LC-ELSD/MS) screening analysis of the walnut phenolic
extracts (Fig. 1
) identified ellagic acid as was previously reported (29
).
In addition, the closely related ellagitannins, valoneic acid dilactone
(negative electrospray ionization: mass 469.4) and pedunculagin (mass
783.7) were observed. There were also three different compounds that
appeared to be ellagitannins on the basis of mass (mass 907.8, 951.9,
1104.0), but their specific identity was not clear. One flavonol, a
quercetin pentoside (mass 433.4) was also observed. These screening
data show that the phenolics in walnut are principally of the
nonflavonoid type and fall into the category of ellagitannins. However,
the specific identities of all of the major phenolic components remain
to be established.
|
All three concentrations of ellagic acid (50, 100 and 150
µmol/L) significantly inhibited AAPH-mediated
oxidation in plasma by 57, 74 and 75% respectively, compared with
control plasma (Fig. 2
). The two higher concentrations (100 and 150 µmol/L GAE)
of walnut extracts significantly inhibited oxidation by 23 and 31% for
heat extract, and 28 and 35% for cold extract, respectively. These
whole-plasma results correspond well with the AAPH-mediated LDL
oxidation data.
|
). The effect was significantly greater in the Cu-mediated oxidation
system, with lag time increasing an average of 0, 39 and 82% (Fig. 3
B). Purified ellagic acid and catechin were used as control
phenolic compounds and compared with control LDL. The highest
concentration of ellagic acid resulted in 87 and 14% increases in lag
time with AAPH- and Cu-mediated oxidation systems, respectively,
and catechin also significantly increased lag time by 82 and 43%,
respectively. Figure 3
C depicts CD formation during
copper-mediated LDL oxidation using a representative concentration
(1 µmol/L) of ellagic acid, heat and cold walnut extracts
and compared with control LDL.
|
-tocopherol up to 20 min,
regardless of whether it was added immediately before or 10 min after
the initiation of oxidation (Fig. 4A
). The walnut heat extract significantly spared the -tocopherol at
the 20-min time point when added immediately before oxidation, but not
when added 10 min after initiation (Fig. 4
B). No
-tocopherol was detected in the walnut phenolic extracts (data not
shown).
|
DISCUSSION
Walnuts are unique among the edible tree nuts because of their
relatively high content of polyunsaturated linoleic and linolenic fatty
acids. It is this favorable lipid profile of nuts that has previously
been proposed as the mechanism of their apparent antiatherogenic effect
in humans. The fatty acids of walnuts are prone to oxidation
(rancidity) and thus, it is of interest that the pellicle surrounding
the kernel is naturally rich in antioxidant polyphenolics, in addition
to the high content of tocopherols in the kernel itself
(30
, 31
). Walnut pellicles contain a number of nonflavonoid
phenolics, based on early work using liquid and paper chromatography
(32
). At least 10 polyphenols have been reported to be
present in English walnut pellicle extract, including the monomers
ellagic acid, gallic acid and methyl gallate, which when present as
polymers and bound to sugars are known as hydrolyzable tannins, and
comprise the majority of the polyphenolics present
(29
, 32
, 33
). The LC-ELSD/MS data presented here confirm
that the hydrolyzable tannins, and specifically ellagitannins, are the
most abundant phenolic constituents. Minor differences were identified
between the heat and cold extracts with respect to relative peak size.
However, the antioxidant capacities of the two walnut extracts were
very similar. The activity of these substances warrants a more thorough
analysis to determine whether the activity is dependent on just a few
or many of the components, and then to establish their complete
chemical identity.
The extract from a handful of walnuts, one serving of 50 g or
8–9 shelled walnuts, contained 802 mg GAE of total phenols, thus
explaining the astringency often associated with eating raw walnuts.
Pecans have a total phenol content of 500 mg GAE in 50 g of
nuts (34
). Unfortunately, no direct comparison of
polyphenolic content can be made to other nuts because of the lack of
existing data. Walnuts have favorable levels of total phenols relative
to other foods reported to have high phenolic and antioxidant levels,
when compared on a per serving basis. A handful of walnuts has
significantly more phenolics than a glass of apple juice (117 mg in 240
mL or 8-oz glass) (35
), a milk chocolate bar (205 mg in a
1.5 oz or 43-g chocolate bar) (11
), or even a glass of red
wine (372 mg GAE of total phenols in 150 mL or 5-oz glass)
(36
). The number of servings of these other foods that
would have to be consumed to equal the total phenolics in a serving of
walnuts is as follows: 1 serving of walnuts = 2.2 servings red
wine = 3.9 servings of milk chocolate = 4.6 servings of apple
juice. However, the energy content of the foods should also be taken
into account when consideration is given to substituting foods low in
polyphenolics with higher energy polyphenol-rich foods. Expressed
in terms of phenolic nutrient density (in this case GAE/kJ in one
serving), the foods can be ranked in the following order: red wine 3.0
> walnuts 2.5 > apple juice 1.0 
milk chocolate 0.9. In
summary, the overall nutritional quality of walnuts as a whole food is
such that it represents one of the best dietary sources of phenolic
antioxidants, -tocopherol and unsaturated fatty acids, in addition
to other components, that can be easily incorporated into individual
diets.
Antioxidant activities of the extracts and purified compounds were in
close agreement with previous reports of phenolic compounds
(37
–39
). The TEAC data demonstrate that walnut extracts
have more antioxidant activity than -tocopherol and that purified
ellagic and gallic acid (monomeric components of walnuts) have even
greater potency than -tocopherol, as assessed by this method. A
possible explanation for the discrepancy between TEAC values for walnut
extracts and the purified compounds is that the presence of phenolic
compounds as larger complexes of hydrolyzable tannins, very good metal
complexing agents compared with simple ellagic acid, in the extract may
result in a lower overall antioxidant activity in this assay.
Additionally, the complex mixture of phenolics found in the extract may
contain compounds that inherently do not have a TEAC value as high as
the purified ellagic acid, or it is possible that some of the
antioxidant activity is lost due to the extraction methods.
Walnut polyphenolic extracts, at concentrations within the range of
expected physiologic plasma levels of dietary phenolics,
(40
–43
), were compared with ellagic acid in view of its
large contribution to the total polyphenolic content in English walnuts
(Fig. 1)
. Ellagic acid has been studied previously in lipid oxidation
assays by a variety of methods, yielding conflicting results
(9
, 22
, 44
, 45
). Formation of CD during LDL oxidation was
used in the present study because clinical studies report the lag time
of CD to be a measure of LDL oxidation that is associated with coronary
heart disease risk (46
, 47
). Ellagic acid significantly
inhibited AAPH- but not Cu-mediated LDL and plasma oxidation (Fig. 3)
. In contrast, both walnut extracts demonstrated significantly
greater ability to inhibit Cu-mediated LDL oxidation than ellagic
acid. Additionally, the extracts significantly inhibited
AAPH-mediated plasma and LDL oxidation, although to a lesser degree
than purified ellagic acid. This suggests that other phenolics present
in the extracts contribute markedly to the inhibition of
Cu-mediated oxidation, probably due in large part to metal
chelation by the tannins. Thus, walnut extracts containing ellagic acid
monomers, polymeric tannins and other phenolics effectively inhibited
in vitro plasma and LDL oxidation induced by generation of hydroxyl,
peroxyl and superoxide radicals in both metal ion–dependent and
–independent oxidation systems.
The walnut polyphenolics and purified ellagic acid had similar modest
significant in vitro effects on maintaining LDL -tocopherol during
oxidative stress. It can be speculated that ellagic acid may be all or
partly responsible for the observed in vitro effect on LDL
-tocopherol, but this is not confirmed by the present experiments.
Previous in vitro studies have demonstrated the ability of other
selected phenolics to maintain and extend LDL -tocopherol levels and
in some cases regenerate -tocopherol (48
–51
). The
concentration of LDL -tocopherol, despite being quantitatively the
predominant antioxidant, contributes to, but is not the primary
determinant that predicts the resistance of LDL to oxidative stress
(51
–54
). The complex milieu of endogenous antioxidants in
the plasma and LDL, including dietary derived polyphenols, all
contribute to the protection of LDL in vivo.
A recent in vivo study reported that daily consumption of walnuts
significantly lowered total and LDL cholesterol but did not
significantly change LDL -tocopherol levels or the ability to resist
oxidation when initiated ex vivo (17
). However, the
ability of LDL to resist oxidation was preserved by the walnut diet
despite a 14 and 83% enrichment in LDL lipids of polyunsaturated
linoleic acid and -linolenic acid, respectively, which are prone to
oxidation. Even though there was no effect on oxidation susceptibility
of isolated LDL after daily consumption of walnuts, it cannot be ruled
out that in vivo, plasma and LDL lipids have acquired an increased
resistance to free radical damage.
It is likely that polyphenolic compounds are localized in the plasma
and are loosely associated with the LDL surface, rather than
partitioned into the LDL lipid core, due to their combined lipophilic
and hydrophilic properties (55
, 56
). Therefore, measurement
of both plasma and LDL oxidation susceptibility and specific markers of
oxidative damage ex vivo will more closely reflect the physiologic
action of dietary polyphenols and their circulating metabolites
(57
). In vitro results for polyphenolic compounds
generally show protection against LDL oxidation and depletion of
-tocopherol, yet in vivo investigations have produced mixed results,
possibly due to variations in design, methodology and the variability
of polyphenolic content in food sources. However, protective effects of
polyphenols have been observed more consistently in studies in which
the antioxidant capacity of whole plasma or plasma lipid peroxidation
products was measured ex vivo (55
, 58
–64
).
The current in vitro data on polyphenolic-rich walnut extracts used at expected physiologic concentrations, demonstrate their potent antioxidant capacity with plasma and LDL lipids and contribute to the overall understanding of how dietary-derived polyphenolics may participate in the complex antioxidant network. The brief LC-ELSD/MS characterization of the predominant walnut phenolic compounds adds to current knowledge of polyphenolic distribution in foods. In addition to the favorable lipid profile of nuts, their phenolic content must now be considered as a potential contributor to the apparent antiatherogenic effect of nuts. These dual benefits of walnuts can be derived only from a whole food. Thus, the role of nuts as antiatherogenic is strengthened by the addition of in vitro data to existing feeding and epidemiologic studies. Further research is required to examine the effects of whole-walnut consumption on plasma polyphenol levels, plasma and LDL antioxidant capacity and ex vivo resistance to oxidative stress.
ACKNOWLEDGMENTS
The authors thank Jody Randolph for her technical assistance.
FOOTNOTES
1 Presented in preliminary form at Experimental
Biology 2001, March 31–April 4, Orlando, FL [Anderson, K. J.,
Teuber, S., Steinberg, F. M. (2001) Walnut
polyphenolic extracts inhibit in vitro plasma and LDL
oxidation. FASEB J. 15: A607 (abs.)]. 
2 Supported in part by a grant from the California
Walnut Commission.
4 Abbreviations used: AAPH, 2,2'-azobis'(2-amidino
propane) hydrochloride; ABTS,
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; CD, conjugated
diene; GAE, gallic acid equivalents; LC-ELSD/MS, liquid chromatography
electrospray detection mass spectrometry; MDA, malondialdehyde
(1,1,3,3-tetramethoxypropane); TBARS, thiobarbituric acid reacting
substances; TEAC, Trolox equivalent antioxidant capacity.
Manuscript received 18 May 2001. Initial review completed 26 June 2001. Revision accepted 20 August 2001.
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