JFS S: Sensory and Nutritive Qualities of Food

Comparative Studies on the Antioxidant

Activities of Nine Common Food Legumes
Against Copper-Induced Human Low-Density
Lipoprotein Oxidation In Vitro
B.J. XU, S.H. YUAN, AND S.K.C. CHANG

ABSTRACT: Epidemiological studies demonstrated that the consumption of dietary antioxidant was associated
with the prevention of atherosclerosis. The aim of this study was to investigate the antioxidant activities of the
hydrophilic extracts from 9 selected legumes based on copper-induced human LDL oxidation model in vitro. The
antioxidant activities were assessed on the basis of the formation of conjugated dienes (lag time of oxidation) and
thiobarbituric acid reactive substances (TBARS) as the early and later stage markers of LDL oxidation. The results
showed that the extracts of black beans (Phaseolus vulgaris L.), lentils (Lens culinaris), black soybeans (Glycine
max), and red kidney beans (Phaseolus vulgaris L.) had significant (P < 0.05) longer LDL oxidation lag times (128.8,
124.2, 107.7, and 111.1 min, respectively) than the LDL control group (94.9 min). No significant lag-time length-
ening was observed in other tested legume extracts. On the other hand, black beans, lentils, black soybeans, red
kidney beans, and pinto beans exhibited higher antioxidant capacities (Trolox equivalents) than yellow peas, green
peas, chickpea, and yellow soybeans in both LDL-conjugated dienes assay and LDL-TBARS assay. Meanwhile, the an-
tioxidant activities of these legumes against LDL-lipid peroxidation in the above assays were found to correlate very
significantly (P < 0.01) with their phenolic substances, and DPPH radical scavenging activity and ORAC (oxygen rad-
ical absorbance capacity). These results suggest that consuming black beans, lentils, black soybeans, and red kidney
beans may have potential in preventing the development of atherosclerosis from the perspective of inhibiting LDL
oxidation.
Keywords: antioxidant activity, food legumes, LDL oxidation, phenolic substances

Introduction others 2007). Phenolic compounds, such as phenolic acids,

A ntioxidants are believed to play a very important role in the

body defense system against reactive oxygen species (ROS),
which are associated with the development of many chronic and
degenerative diseases, including cancer (Ames and others 1995),
cardiovascular disease (Diaz and others 1997; Uchi 2000), and neu-
ronal degeneration such as Alzheimer’s and Parkinson’s disease
(Shahidi 1997) and aging (Ames and others 1993). The epidemio-
logical studies have shown positive correlations between the con-
sumption of foods with a high content of phenolic substances in
plant foods and high antioxidant values with decreasing incidence
of several diseases (Kushi and others 1999; Kris-Etherton and others
2002). Legume foods possess potential health benefits with respect
to cardiovascular disease protection (Anderson and others 1999;
Kushi and others 1999) and breast cancer prevention (Adebamowo
and others 2005). Legumes are a rich source of polyphenols,
which have high antioxidant activities (Cardador-Martinez and
others 2002; Troszynska and others 2002; Amarowicz and others
2003; Beninger and Hosfield 2003; Madhujith and others 2004;
S: Sensory & Nutritive Qualities of Food
flavonols, flavones, isoflavones, anthocyanins, and condensed tan-
nins, have been identified and characterized in food legumes
(Champ 2002; Chang 2002; Beninger and Hosfield 2003; Madhujith
and others 2004; Xu and Chang 2007; Xu and others 2007).
Physiologically, low-density lipoprotein (LDL) is used to trans-
port cholesterol in the body and its oxidation plays a key role in the
atherosclerosis development process (Steinberg and others 1989;
Grundy 1995). It has been proposed that among all in vitro antiox-
idant capacity assays, measuring LDL antioxidant activity is more
physiopathologically important and more informative for screen-
ing antioxidant activity of foods for preventing atherosclerosis than
other methods (Katsube and others 2004). Freshly prepared or com-
mercial LDL has been widely used to analyze the antioxidant ac-
tivity of pure compounds or crude extracts of natural products
and daily foods (Esterbauer and others 1992; Daughty and Roselaar
1995; Kerry and Abbey 1998; Iwai and others 2002; Takahashi and
others 2005). The most widely used method for measuring the re-
sistance of LDL to oxidation in vitro is by determining the lag

Heimler and others 2005; Madhujith and Shahidi 2005; Xu and
MS 20070233 Submitted 4/2/2007, Accepted 6/13/2007. Authors Xu, Yuan,
and Chang are with Dept. of Cereal and Food Sciences, North Dakota State
Univ., Fargo, ND 58105, U.S.A. Author Xu is also with The Pharmaceutical
Inst., Dalian Univ., Dalian 116622, China. Direct inquiries to author Chang
(E-mail: kow.chang@ndsu.edu).
time for conjugated diene formation or thiobarbituric acid reac-
tive substances (TBARS) in response to lipid peroxidation mediated
by Cu2+ ions (Sattler and others 1998; Iwai and others 2002; Jeong
and others 2004; Katsube and others 2004; Takahashi and others
2005).
There are many different varieties/types of food legumes in
the world. We have recently analyzed phenolic substances (total

S522 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 7, 2007 

C 2007 Institute of Food Technologists
doi: 10.1111/j.1750-3841.2007.00464.x
Further reproduction without permission is prohibited
Comparative studies on the antioxidant activities . . .
phenolic content, flavonoids, condensed tannins) and antioxidant
properties of 33 types of cool season legume materials [peas (Pisum
sativum L.), lentil (Lens culinaris), and chickpea (Cicer arietinum
L.)], and compared to several major classes of legumes, including
food-grade soybeans (Glycine max) and common beans (Phaseo-
lus vulgaris), and showed that the hydrophilic extracts of legumes
have significantly different phenolic compositions (Xu and others
2007), which require different solvents for extractions (Xu and
Chang 2007). We also showed that the phenolic compositions in the
legume hydrophilic extracts are strongly correlated to their antioxi-
dant activities as measured by 3 different assay methods, including
DPPH, FRAP, and ORAC tests. Lentils, colored common beans, and
soybeans possessed high antioxidant capacities.
There are only 3 literature reports regarding the antioxidant ac-
tivity of legumes against human LDL oxidation, two on soybeans
(Hwang and others 2001; Takahashi and others 2005), and another
one on common beans (Madhujith and Shahidi 2005). In addi-
tion, 1 study published the rat LDL oxidation inhibition of fer-
mented natto soybeans (Iwai and others 2002). The cool season
food legumes (CSFL), including green and yellow peas, lentils, and
chickpeas, have not been reported with respect to their anti-LDL
oxidation capacity. Due to differences in the methods and materi-
als, comparisons of the results between interlaboratory reports are
impossible. Therefore, the 1st specific aim of this study was to in-
vestigate the antioxidant effects of the hydrophilic extracts of 4 se-
lected CSFL against human LDL oxidation and compare to that of
5 other commonly consumed food legumes. The 2nd objective was
to analyze the statistical correlations between the inhibition of LDL
oxidation obtained from this study and other antioxidant activities
and phenolic compositions in legumes using the results published
in our previous articles.
Materials and Methods
Legume samples
Several typical legume varieties were selected. Red Chief lentils
(Lens culinaris) were purchased from Spokane Seed Co. (Spokane,
Wash., U.S.A.), black turtle beans (Phaseolus vulgaris L.) were pro-
vided by Univ. of Idaho Foundation Seed Program Kimberly Re-
search & Extension Center (Kimberly, Idaho, U.S.A.). Red kidney
beans (Phaseolus vulgaris L.) and pinto beans (Phaseolus vulgaris
L.) were purchased from the Red River Commodity (Fargo, N.Dak.,
U.S.A.). Small chickpeas (Cicer arietinum) were provided by Agri-
care United (Ray, N.Dak., U.S.A.). Black soybeans (Glycine max)
were purchased from Hsingchu Farm Coop. in Taiwan. Proto soy-
beans (Glycine max), which were bred for soymilk and tofu mak-
ing, were obtained from Sinner Brothers and Bresnahan (Casselton,
N.Dak., U.S.A.). SW Capri yellow peas (Pisum sativum L.) were
purchased from Meridian Seeds LLC (West Fargo, N.Dak., U.S.A.).
Cruiser green peas (Pisum sativum L.) were obtained from Idaho
(US Dry Pea and Lentil Assn.). Before processing, beans were hand
sorted to remove splits, small wrinkled beans, and foreign materi-
als. Moisture content was determined by drying the sample in an
air-circulated oven at 110 ◦ C until a constant weight was obtained
(AOAC 2000). The results of antioxidants activities were expressed
on dry weight basis.
Extraction of legume samples
Natural-dried legumes were ground to a powder with an IKA

all basic mill (IKA Works Inc., Wilmington, N.C., U.S.A.) and sub-
sequently sieved with a 60-mesh screen. The legume powder was
accurately weighed in 0.5-g aliquots, then 5 mL of acetone/water
(50:50, v/v) extraction solvent was added to peas, chickpeas, and
soybeans, and 5 mL of acetone/water/acetic acid (70:29.5:0.5,
v/v/v) extraction solvent was added to lentils, black soybeans, and
common beans. The reason for selecting these specific solvents sys-
tems for the 2 respective legume groups was based on a prelimi-
nary study (Xu and Chang 2007) that these respective solvents gave
the best yields of phenolic contents and antioxidant activity. The
mixtures were shaken at 300 rpm at room temperature on an or-
bital shaker for 3 h. The mixtures were extracted for another 12 h
in the dark. The extracts were centrifuged at 3000 rpm for 10 min,
and the supernatants were removed into new tubes. The precipi-
tated residues were re-extracted with 5 mL of respective solvents.
The 2 extracts from both extractions were combined and stored at
4 ◦ C in the dark until analyses. Extractions were performed in tripli-
cate for each individual legume. Analyses were performed on each
individual extract.
Human LDL isolation and
preparation of EDTA-free LDL
LDL (1.063 > d > 1.019 g/mL, 15 ◦ C) was isolated by sequen-
tial ultracentrifugation (Havel and others 1955). Human blood
plasma (280 mL) from an individual normolipidemic healthy donor
was obtained from the United Blood Service (UBS, Fargo, N.Dak.,
U.S.A.). Before analysis, plasma containing EDTA (density approx-
imately 1.011 g/mL) was adjusted to d = 1.019 g/mL using a d =
1.346 g/mL of KBr/NaCl solution and saline. Ultracentrifugation
was then carried out for 3 h at 15 ◦ C with a Beckman L5-50 ultra-
centrifuge and an SW 41 Ti rotor at 39000 rpm (187728 g). Chy-
lomicron, very low-density lipoprotein (VLDL), and intermediate-
density lipoprotein (IDL) forming a distinct layer on the top were
removed by aspiration. The infranate was transferred to a new tube
and adjusted to d = 1.063 g/mL. LDL was then separated by ul-
tracentrifugation using the same conditions (41 Ti, 187,728 g) at
15 ◦ C for 12 h. The LDL fraction was collected from the top by as-
piration and then dialyzed at 4 ◦ C in the dark using Spectra/Por 2
dialysis membranes against 4 exchanges of PBS (pH 7.4) containing
0.1% EDTA. The protein concentration of freshly prepared, dialyzed
LDL was determined according to the Bicinchoninic acid method
(Smith and others 1985; Chang 2003) using bovine serum albumin
(BSA) as standards. Three milliliters of aliquot of dialyzed solution
were stored as a stock solution at –80 ◦ C in the dark until use. Prior
to LDL oxidation assay, an aliquot of LDL storage was thawed, then
dialyzed in a 400-fold volume of 100 mM PBS at pH 7.4 containing
160 mM NaCl at 4 ◦ C in the dark overnight. The dialyzed LDL was
diluted to 50 µg/mL by using 100 mM PBS at pH 7.4 contained 160
mM NaCl and divided into 30 µL aliquots, which were covered with
tinfoil to protect from light exposure, and frozen at –80 ◦ C before
use.
Copper-induced oxidation of LDL
EDTA-free LDL (50 µg/mL) was oxidized with 10 µM CuSO 4 and
then stopped by the addition of 1 mM EDTA and 0.5 mM BHT (Kerry
and Abbey 1998). In our experiments, oxidation was carried out in
the presence of diluted legume extracts. After the incubation, con-
jugated dienes and TBARS formation were measured as described
subsequently.
S: Sensory & Nutritive Qualities of Food
Detection of conjugated dienes
The detection of conjugated dienes (early oxidation products)
of LDL oxidation induced by copper ion was performed by the
method of Kerry and Abbey (1998) with slight modifications. Briefly,
1 mL of an LDL solution (50 µg/mL) in PBS was incubated with di-
luted legume extracts. The control experiments consisted of identi-
cal assay conditions but without adding legume sample. Oxidation
Vol. 72, Nr. 7, 2007—JOURNAL OF FOOD SCIENCE S523
 Comparative studies on the antioxidant activities . . .

was initiated immediately after adding 10 µM final concentration of Statistical analysis

CuSO 4 ·5H 2 O dissolved in deionized distilled water. The formation
of conjugated dienes was measured by monitoring the absorbance
at 234 nm using a double-beam spectrophotometer (Model UV 160,
Shimadzu, Kyoto, Japan) equipped with accessories for multichan-
nel kinetic analysis (CPS-240A, Shimadzu, Kyoto, Japan). The ref-
erence cuvette contained an equal volume of LDL for subtract-
ing instrumental drift and LDL background absorbance from the
samples. Oxidation kinetics were recorded every 10 min for 4 h at
37 ◦ C. The plot of absorbance against time produced 3 phases: (1) a
lag phase, (2) a propagation phase, and (3) a decomposition phase.
The lag time was defined as the intercept between the tangent of
the absorbance curve during the propagation phase and the base-
line, and was calculated by imputing y = 0 into linear fit equa-
tion of propagation phase absorbance to obtain x value (lag time in
minutes) in the time axis (X -axis). The LDL-conjugated dienes in-
hibitory activities were expressed as micromoles of Trolox equiva-
lent (TE) per gram legume on dry weight basis. Meanwhile, the area
under the curve of the absorbance increase from 0 to 4 h (AUC 0–4h )
was also calculated.
LDL-TBARS assay
The LDL oxidation was determined spectrophotometrically by
measuring the amount of TBARS (Liu and Ng 2000). Briefly, 1 mL of
an LDL solution (50 µg/mL) in PBS was incubated with 10 µM final
concentration Cu2+ in the presence or absence (control) of 20 µL of
diluted legume extracts. The oxidation was performed in a screw-
capped 10 mL test tube at 37 ◦ C in a shaking water bath for 3 h in
the dark. Oxidation reaction was stopped by adding 1 mM EDTA (fi-
nal concentration). One milliliter of 20% (w/v) trichloroacetic acid
(TCA) and 1 mL of 0.67% (w/v) thiobarbituric acid (TBA) in 0.2 M
NaOH were added to the post-incubation mixture. The mixture
was heated at 95 ◦ C for 30 min and cooled. After centrifugation at
1500 × g for 15 min to remove precipitated proteins, the ab-
sorbance of the supernatant was measured at 532 nm. Lipid perox-
idation inhibitory ratio was calculated using the following formula:
Inhibitory ratio (%) = (Ac − As)/Ac × 100
where Ac was the absorbance of the LDL control and As was the
absorbance of the tested legume samples. The antioxidant capacity
of legumes was expressed as micromoles of Trolox equivalent (TE)
per gram through the calibration curve of Trolox. Linearity range of
the calibration cure was 20 to 50 µM (r = 0.98).
Statistical analysis was performed with analysis of variance
(ANOVA) followed by Dunnett’s post hoc test for multiple compar-
isons among the groups. Differences were considered to be statisti-
cally significant if the probability value was less than 0.05 (P < 0.05).
A Pearson correlation test was conducted to determine the correla-
tions between variables, including LDL-inhibition, total phenolic
content, and DPPH and ORAC values published in our recent arti-
cle using the same legume materials (Xu and others 2007).
Results and Discussion
Inhibition of in vitro copper-induced
oxidation of LDL
Currently, the formation of conjugated dienes, measured at
234 nm, is considered the most appropriate marker of LDL oxida-
tion (Sattler and others 1998). The formation of conjugated dienes
represents an early stage in the oxidation process while TBARS for-
mation represents the oxidation products at a late stage. Therefore,
the formation of conjugated dienes as well as TBARS was measured
in this study. The 1st experiments involved determining an appro-
priate dilution of legume extract, which could be used as a starting
point for comparing the relative antioxidant activities of different
legumes. Legume extracts were diluted using PBS buffer to the fol-
lowing dilutions: 1 in 5, 1 in 10, and 1 in 50. These diluted solutions
as well as the original extracts were used as test samples; to explore
appropriate dilutions for different legumes. The dilution effect of
yellow peas as a typical example was performed in our preliminary
experiments. The results showed that original solution could not be
directly used as test sample; however, due to no significant lag-time
variation in comparison to the LDL control, over-dilution (1 in 10 in
the case of yellow peas) also did not work for comparing lag times.
Suitable dilutions had to be worked out to observe the LDL oxida-
tion inhibition effect. After preliminary trials, dilution selected for
later experiments was 1 in 5 for yellow peas, green peas, chickpeas,
and yellow soybeans, and 1 in 50 for black beans, lentils, black soy-
beans, red kidney beans, and pinto beans. At these respective di-
lutions, the experiments resulted in an increase in lag time of con-
jugated diene formation as compared to that for the control LDL
oxidation.
Triplicate measurements of lag time were performed on each
of the triplicate extracts for each individual legume. The mean lag
times are summarized in Table 1 and the kinetic plots of 2 selected
legumes with different reaction rates as well as LDL control and

Table 1 --- Antioxidant effects of selected common legumes against copper-mediated LDL oxidation based on detec-
tion of conjugated dienesa
Selected Concentration Lag time LDL-CD AUC 0–4h
legumes of test sampleb (min) (µmol TE/g)c of CD increase
LDL control 50 µg/mL 94.92 ± 1.09 --- 74.82 ± 0.91
Yellow pea 10 mg/mL 97.49 ± 2.71 1.58 ± 0.11f 76.32 ± 3.67
Green pea 10 mg/mL 94.28 ± 13.08 1.50 ± 0.49f 82.29 ± 2.89d
Chickpea 10 mg/mL 90.76 ± 0.11 1.23 ± 0.01f 76.65 ± 1.57
Yellow soybean 10 mg/mL 84.47 ± 1.58d 0.94 ± 0.07f 79.60 ± 0.66d
Black bean 1 mg/mL 128.87 ± 7.42d 29.43 ± 0.85a 58.03 ± 3.80d
S: Sensory & Nutritive Qualities of Food

Lentil 1 mg/mL 124.18 ± 0.93d 28.37 ± 0.45b 67.70 ± 0.95d

Black soybean 1 mg/mL 107.73 ± 1.48d 19.43 ± 0.49d 73.73 ± 0.55
Red kidney 1 mg/mL 111.11 ± 1.26d 22.55 ± 0.59c 65.72 ± 0.57d
Pinto bean 1 mg/mL 95.63 ± 0.43 14.98 ± 0.22e 74.82 ± 0.91
50 µm Trolox 50 µm 165.88 ± 2.29d --- 34.76 ± 0.14d
a
Assays were performed in triplicate and data were expressed as mean ± standard deviation of 3 separate determinations. Control incubations were carried out in
the presence of phosphate-buffered saline. b Concentration of test sample was expressed as mg legume/mL. c The LDL-conjugated dienes inhibitory activities were
expressed as micromoles of Trolox equivalent (TE) per gram legume on dry weight basis, means followed by different letters are significantly different (P < 0.05).
d
The mean difference is significant at the 0.05 level, Dunnett t -test was performed by comparing sample groups against LDL control group. Trolox was used as
positive control.

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Comparative studies on the antioxidant activities . . .

positive control (Trolox) against LDL oxidation are illustrated in
Figure 1. The extracts from black beans, lentils, black soybeans,
and red kidney beans at the concentration of 1 mg legume/mL
produced a significantly longer LDL oxidation lag time (128.87,
124.18, 107.73, and 111.11 min, respectively) than the LDL control
(94.92 min). No significant lag-time lengthening was observed in
the assays of other legume extracts. In addition, the extracts from
black beans, lentils, black soybean, and red kidney beans at the
concentration of 1 mg legume/mL had a longer LDL oxidation lag
time than yellow peas, green peas, chickpeas, and yellow soybeans
at the concentration of 10 mg legume/mL. This indicated that black
beans, lentils, black soybean, and red kidney beans at the concen-
tration of 10 mg legume/mL would have much longer lag times
than the current values. Due to the use of different concentrations
of diluted test samples and different concentrations of LDL and
copper ion, a direct comparison on lag times with that reported
in the literature was impossible. However, our results had consis-
tent tendency with the findings of Takahashi and others (2005), who
reported that black soybeans had a longer LDL oxidation lag time
(205 ± 16 min) than yellow soybeans (65 ± 3 min).
In addition to lag time as a marker, antioxidant activities of
legumes against LDL oxidation were reflected by comparing the
area under the curve of the absorbance increase of the conjugated
dienes from 0 to 4 h (AUC 0–4h ). The AUC method included a time
factor in the measurement of the progress of the oxidation of lipids
and therefore providing additional information beyond lag time as
a marker. The peak and shape of the curves differed slightly among
samples and control. The AUC 0–4h values of all legume extracts are
presented in Table 1. The extracts from black beans, lentils, and red
kidney beans at the concentration of 1 mg legume/mL had signif-
icantly lower AUC 0–4h values (58.03, 67.70, and 65.72, respectively)
than that of LDL control (74.82). However, no significant AUC 0–4h
value decreases were observed in the tests of yellow peas, chick-
peas, black soybeans, and pinto beans. Interestingly, green peas
and yellow soybeans exhibited significant higher AUC 0–4h value as
compared to LDL control; these may be partly due to the shortening
of their lag-times and the slight decreases of the absorbance dur-
ing the decomposition phase as compared to LDL control, finally
contributing to the increases of the areas under the curves. The
0.8
LDL control
Lentils
Black soybeans
50um Trolox
0.6
Absorbance at 234 nm
slower decomposition of conjugated dienes in the legumes samples
might have contributed to the antioxidant effect as measured by
LDL-TBARS (Table 2). Judging from the kinetic absorbance plot, the
decreases of AUC 0–4h values of black beans, lentils, and red kidney
beans might be due to the lengthening of their lag times. These re-
sults further validated the antioxidant effects of black beans, lentils,
and red kidney beans against LDL oxidation. In addition, for com-
parison purpose, antioxidant capacity of 50 µM of Trolox (a posi-
tive antioxidant standard) was also examined. The results showed
that 50 µM of Trolox exhibited longer lag time and lower AUC 0–4h
values (Table 1, Figure 1) than all tested legume samples. There-
fore, to investigate potential relationships between antioxidant ac-
tivities against LDL oxidation (based on lag time) and concentra-
tions of legume samples, further experiments using variable Trolox
concentration standards (10 to 100 µM) were carried out. A no-
table dose-dependent relationship (quadratic fit equation was y =
54.036 + 2.926x – 0.014x2 ) between lag times and concentrations of
Trolox was found. However, the dose-dependent relationship was
not linear within all concentrations. The linear range was from 10
to 50 µM, which corresponded to approximately 84 to 165 min lag
times. These results indicated that the lag times representing the
antioxidant activities of legume samples against LDL oxidation fell
within the linear range of the dose–response curve of Trolox. There-
fore, LDL-conjugated dienes inhibitory activities were calculated
as micromoles of Trolox equivalent through the linear range (from
10 to 50 µM) of the curve of Trolox. The results are presented in
Table 1. Significant (P < 0.05) differences in LDL-conjugated di-
enes inhibitory activity existed among most legumes. No signifi-
cant differences were observed among peas, chickpea, and yellow
soybeans.
Since TBARS formation had been used as a marker of lipid per-
oxidation, LDL-TBARS assay was performed to measure inhibitory
activities of legume extracts on the formation of the late-stage prod-
ucts of LDL oxidation. The results are presented in Table 2. Sig-
nificant (P < 0.05) differences in TBARS inhibitory activity (Trolox
equivalents) existed among some legumes. No significant differ-
ences were observed among black beans, lentils, red kidney, and
pinto beans. The group of black beans, lentils, black soybeans,
red kidney beans, and pinto beans exhibited a higher antioxi-
dant capacity (5.7 to 6.4 µmol TE/g) than the group of yellow
and green peas, chickpea, and yellow soybeans (2.1 to 2.4 µmol
TE/g). Both methods of detecting LDL oxidation, LDL-conjugated
dienes-Trolox and LDL-TBARS inhibition, exhibited consistent ten-
dency and were highly correlated (r = 0.94, P < 0.001) with each
other. In the literature, many oxidation markers have been used for

Table 2 --- Antioxidant effects of selected common

0.4
legumes against copper-mediated LDL oxidation based
on LDL-TBARS assaya
Selected legumes LDL-TBARSb (µmol TE/g)
0.2
Yellow pea 2.35 ± 0.02c
Green pea 2.09 ± 0.08d
Chickpea 2.19 ± 0.12cd
0.0 Yellow soybean 2.20 ± 0.10cd
S: Sensory & Nutritive Qualities of Food

0 50 100 150 200 250 Black bean 6.39 ± 0.08a

Time (min) Lentil 6.24 ± 0.18a
Black soybean 5.72 ± 0.23b
Figure 1 --- Kinetic curves of legume extracts against Cu2+ - Red kidney 6.40 ± 0.10a
mediated LDL oxidation. LDL (50 µg/mL) was incubated Pinto bean 6.35 ± 0.07a
with 10 µM of CuSO 4 in the presence or absence (LDL a
The TBARS inhibitory activities were expressed as micromoles of Trolox
control) of legume extract. Conjugated diene formation equivalent (TE) per gram legume on dry weight basis. Data are expressed as
was measured by determining the absorbance at 234 nm mean ± standard deviation (n = 3). Means followed by different letters are
every 10 min for 4 h. Trolox was used as a reference an- significantly different (P < 0.05). b TBARS means thiobarbituric acid reactive
tioxidant. substances produced due to LDL peroxidation.

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 Comparative studies on the antioxidant activities . . .
monitoring lipid oxidation, our study using multiple measures of idant activities in different pH values. Furthermore, the mixture of
lag time, CD-Trolox equivalent, and AUC (area under the curve), phenolic substances in the bean extract may have a synergistic ef-
and TBARS inhibition provided a good overall picture of antioxi- fect, which is affected by varying test conditions.
dant characteristics of food legumes.
Correlations between LDL oxidation and other
antioxidant assay values and phenolic compositions
Since we had analyzed and reported significant differences in
phenolic substance compositions and antioxidant activities (DPPH
and ORAC) in the extracts from these 9 legume materials in our pre-
vious report (Xu and others 2007), it was of interest to understand
the correlations between these variables. Therefore, linear regres-
sion analyses were performed to investigate correlations among
antioxidant assays and compositions (between LDL-TBARS inhibi-
tions or CD-Trolox and DPPH free radical scavenging values, ORAC,
and phenolic substance contents). A higher correlation coefficient
(r = 0.97, P < 0.01) existed between the DPPH radical scavenging
activity and the LDL-TBARS values, while a relatively lower corre-
lation coefficient (r = 0.81, P < 0.001) existed between the ORAC
assay and the LDL-TBARS values. The CD-Trolox assay also was
highly correlated with the DPPH and ORAC assay (r = 0.97 and 0.83,
respectively, P < 0.001). Different antioxidant test methods utilize
different reaction mechanisms (Prior and others 2005). The ORAC
test reaction in removing reactive oxygen species utilizes the hy-
drogen transfer mechanism whereas the DPPH utilizes the electron
transfer mechanism. The higher correlations between the anti-LDL
oxidation and DPPH assay values suggested that the anti-oxidation
mechanisms in these 2 antioxidant tests by the legume materials
might be similar.
Regarding the correlations between LDL-antioxidant activity
and phenolic substances, the highest correlation coefficient (r =
0.94, P < 0.001) existed between the LDL-TBARS inhibition assay
and the total flavonoid content, followed by that (r = 0.87, P <
0.001) between the LDL-TBARS assay and the total phenolic con-
tent, and that (r = 0.79, P < 0.001) between the LDL-TBARS and
condensed tannins content. The correlations between conjugated
dienes-Trolox (CD-Trolox) values of legumes and phenolic com-
pounds also showed highly significant correlations between CD-
Trolox and phenolic substances (r = 0.96 for total phenolic con-
tent, 0.93 for total flavonoids, and 0.89 for condensed tannins, P <
0.001). These results indicated that total flavonoids or condensed
tannins played a possible role in the overall antioxidant activity
of legumes, especially in lentils, black beans, red kidney beans,
and pinto beans. Our previous research showed high correlations
between antioxidant methods (DPPH, ORAC, and FRAP) and be-
tween antioxidant activities and phenolic compositions in legume
extracts (Xu and others 2007). Different phenolic substances had
different degrees of contributions to the overall antioxidant activi-
ties of different legume varieties/materials.
Differences in the composition and structures of phenolic sub-
stances in different types of legumes also had been reported by
other researchers. Black soybean seed coat had been reported to
contain much more anthocyanidin pigment than the yellow soy-
bean seed coat, which had a much lower LDL-oxidation inhibi-
tion than black soybean (Takahashi and others 2005). Aglycones of
S: Sensory & Nutritive Qualities of Food
isoflavones in the soy cotyledons also had higher oxidation inhibi-
tion rate (Takahashi and others 2005). Anthocyanidins, procyani-
dins, and phenolic acids were reported to be the major phenolic
compounds in dry beans (Madhujith and others 2004). Madhujith
and Shahidi (2005) compared the LDL-oxidation inhibition by 3
color common beans and stated that the differences in anti-LDL
oxidation activities were due to the composition and structural dif-
ferences of phenolic compounds, which may have different antiox-
S526 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 7, 2007
Conclusions
C onsidering the results using the selected antioxidant activity
assays, the most powerful antioxidants were lentils, colored
common beans, and black soybeans. With powerful antioxidant ac-
tivities against LDL oxidation and free radicals, lentils seem to be
the best among all tested food legumes for the development of a
dietary supplement for promoting heart health and for prevent-
ing cancers. Aside from lentils, black beans, black soybeans, and
red kidney beans possess potent antioxidant effect in the protec-
tion of LDL oxidation as induced by copper ion in vitro. Further
investigations on antioxidant, anticancer, and anti-atherosclerotic
effects using cellular and animal models also should be conducted
to compare the health promoting effects of selected food legumes
to antioxidant-rich fruits, and to understand how the effects are in-
fluenced by food processing, which is necessary for converting the
raw legume materials to palatable foods.
Acknowledgments
USDA-CSREES Cool Season Legumes Programs administered
through the Univ. of Idaho (Grant nr FAR100773), North Dakota
Soybean Council, USDA-CSREES-NRI CGP #2006-00907 supported
this research. The authors thank Dr. Steven Meinhadt in the Dept.
of Plant Pathology in the use of ultracentrifuge, and Fargo Branch of
the United Blood Service (UBS) for providing human blood plasma.
We would also like to thank Eric Bartch of the North Dakota Dry Pea
& Lentil Assn., and Todd Scholz of U.S. Dry Pea & Lentil Assn., and
mentioned companies for providing legume samples.
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