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Pharmaceutical Biology 1388-0209/01/3904-300$16.00
2001, Vol. 39, No. 4, pp. 300–304 © Swets & Zeitlinger
Evaluation of the Antioxidant Activity of Legumes
Chun-Ching Lin 1 , Sue-Jing Wu 2 , Juei-Shen Wang 2 , Jenq-Jer Yang 3 and Cheng-Hsiung Chang 3
1
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China; 2 Department
of Health and Nutrition, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan, Republic of China; 3 Department of
Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan, Republic of China
Abstract
The anti-lipid peroxidation activity, free radical scavenger
activity and anti-superoxide formation of hot water extracts
of legumes (HWEL), such as mung bean (Phaseolus radiatus
L.), adzuki bean (Phaseolus aureus Roxb.), black bean
(Glycine max (L.) Merr.) and rice bean (Phaseolus calcaratus
Roxb.), were evaluated to test antioxidant activity.
The results showed that all HWEL exhibited remarkable inhibition
of FeCl 2 -ascorbic acid induced lipid peroxidation
of mouse liver homogenate. All extracts showed anti-lipid
peroxidation activities. Both mung bean and adzuki bean
extracts demonstrated the strongest anti-lipid peroxidation
activity and the highest superoxide anion scavenging activity.
All extracts of legumes showed anti-superoxide formation
activity. Treatment with black bean extracts exhibited the
most powerful activity. Therefore, a component of the diet,
HWEL, is a potential antioxidant.
Keywords: Phaseolus radiatus L., Phaseolus aureus Roxb.,
Glycine max (L.) Merr., Phaseolus calcaratus Roxb., antilipid
peroxidation, free radical scavenger activity, antioxidant,
superoxide anion.
Introduction
Lipid peroxidation not only lowers the nutritive value of food
and deteriorates the taste and flavor, but may contribute
to aging, coronary heart disease, diabetes mellitus, stroke,
rheumatic disease, various liver disorders and carcinogenesis
(Slater & Cheeseman, 1987; Addis & Warner, 1991;
Wong et al., 1987). It has been reported that reactive oxygen
-
species (ROS) such as ·O 2 (superoxide anion), ·OH
(hydroxyl radical), H 2 O 2 (hydrogen peroxide) and ·O 2 (singlet
oxygen), induce cellular injury and initiate peroxidation
of polyunsaturated fatty acids in biological membranes
(Compori, 1985; Halliwell, 1997).
a-Tocopherol, a natural antioxidant, is an effective antioxidant
for lipid-containing foods, but it has limited usage
(Osawa & Namiki, 1981). Antioxidative substances obtained
from plant sources, such as fruits, vegetables, spices, tea,
grains, leaf, and root have been described (Namiki, 1990;
Kikuzaki & Nakatani, 1993; Yen & Chen, 1995; Giese, 1996;
Wang et al., 1996; Oomah & Mazza, 1996; Richheimer
et al., 1996; Lin et al., 1997; Duh, 1998).
Leguminous seeds such as mung bean (Phaseolus radiatus
L.), adzuki bean (Phaseolus aureus Roxb.), black bean
(Glycine max (L.) Merr.) and rice bean (Phaseolus calcaratus
Roxb.), are widely distributed in tropical and subtropical
beaches and native to southern Taiwan (Tainan, Kaohsiung,
Pintung). They have been regarded as folk medicines and
foods or beverages in the daily diet, and widely used in the
treatment of antidote, edema, diuretic, antifebrile, carminative,
etc. (Perry, 1980). Legumes can be utilized as valuable
ingredients of various products. Generally, beverages are prepared
from hot water extracts of legumes (HWEL). However,
it remains unclear if HWEL possess antioxidant activity.
Therefore, this study was undertaken to evaluate the antioxidant
activity of HWEL and to clarify the mechanism of
action.
Materials and methods
Materials
Samples of mung bean (Phaseolus radiatus), adzuki bean
(Phaseolus aureus), and black bean (Glycine max) were
obtained from Tainan District Agriculture Improvement
Station, Taiwan, while that of rice bean (Phaseolus calcaratus)
was purchased from a Chinese herb store in Tainan.
Accepted: January 15, 2001
Address correspondence to: Chun-Ching Lin, Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan,
Republic of China; Tel: 886-7-3121101 ext. 2122; Fax: 886-7-3135215; E-mail: aalin@ms24.hinet.net

Antioxidant activity of legumes 301
Chemicals
L-(+)-Ascorbic acid (AA), thiobarbituric acid (TBA), xanthine,
xanthine oxidase, cytochrome c and a-tocopherol were
purchased from Sigma Chemical Co. (St. Louis, MO).
Preparation of extracts
Each sample (100g) was extracted with 1L of boiling water
for 1 h. The extracts were filtered; the residue was reextracted
under the same conditions, and the combined
filtrates were evaporated to dryness under vacuum and the
yield of soluble constituents (mung bean, adzuki bean, black
bean and rice bean ) were 18.76, 21.80, 15.64 and 24.14%,
respectively.
Animals
Male ICR mice (6 weeks old) were obtained from the animal
center, National Cheng Kung University, Tainan.They were
housed in an air-conditioned room at 22 ± 3°C, 55 ± 5%
humidity, and fed a standard laboratory diet and tap water
throughout the investigation. The mice were used for FeCl 2 -
ascorbic acid-induced lipid peroxidation.
FeCl 2 -ascorbic acid stimulated lipid peroxidation in
mouse liver homogenate
The effect of crude extracts on mouse liver homogenate
induced with FeCl 2 -ascorbic acid and lipid peroxidation was
determined by the method of Yoshiyuki et al. (1981) and
Wong et al. (1987). A mixture containing 0.5 mL of liver
homogenate, 0.1 mL of Tris-HCl buffer (pH 7.2), 0.05 mL of
0.1 mM ascorbic acid, 0.05 mL of 4 mM FeCl 2 and 0.05mL
of various concentrations of crude extracts, or a-tocopherol,
were incubated for 1 h at 37°C. After incubation, 9 mL of distilled
water and 2 mL of 0.6% TBA were added and then
shaken vigorously. The mixture was heated for 30 min in a
boiling water bath (100°C). After cooling, 5 mL of n-butanol
were added and the mixture was shaken vigorously. The
n-butanol layer was separated by centrifugation at 1000 ¥ g
for 10 min. The absorbance of the supernatant was read at
532 nm against a blank, which contained all reagents expect
liver homogenate. The protein content was determined by
the method of Lowry et al. (1951).
Cytochrome c test
Enzymatic formation of superoxide anions was assayed by
reduction of cytochrome c as described by McCord and
Fridovich (1969). Samples were dissolved in distilled water
to 10 mg/mL, diluted with distilled water to various concentrations,
and added to a solution containing 0.07 units/mL of
xanthine oxidase, 100mM xanthine, and 50 mM cytochrome
c. Following a 3min incubation at the room temperature,
absorption was read at 550 nm.
Xanthine oxidase inhibition test
Xanthine oxidase activity was estimated by the formation of
uric acid from xanthine (Chang et al., 1994). Samples were
dissolved in distilled water to 15mg/mL, and diluted with
50mM KH 2 PO 4 buffer (pH 7.8) to obtain each concentration,
and added to a solution contained 100 mM xanthine in phosphate
buffer with 20 mL of xanthine oxidase (0.4 units). Following
a 3min incubation at room temperature, superoxide
formation was determined by measuring uric acid production
by spectrophotometry at 295 nm. IC 50 values of each sample
was calculated.
Statistical analysis
Statistical analysis involved use of the Statistical
Analysis System (SAS) software package. The data
were indicated as the mean ± SD. Analysis of variance was
performed by one-way analysis of variance (ANOVA)
procedures. Significant differences between the inhibitory
rates were determined by Duncan’s Multiple Range tests
(Duncan, 1957).
Results
Anti-lipid peroxidation activity
The inhibitory effect of different concentrations of HWEL
on malondialdehyde (MDA) production in mouse liver
homogenate, induced by FeCl 2 -ascorbic acid in vitro,
are shown in Table 1. Inhibition of MDA formation
increased with increasing concentrations of HWEL. All
extracts, in the concentration range of 0.3–3.0mg/mL,
showed anti-lipid peroxidation activities, and the inhibition
rates were in the range of 51–100%. Significant differences
(P < 0.05) were found between the different concentrations
of hot water extracts in various legumes. The 50%
inhibitory concentration (IC 50 ) values ranged from
0.15–0.26 mg/mL in the thiobarbituric acid test. These
results indicate that legumes displayed remarkable antioxidant
activity.
Free radical scavenger activity
Enzymatic formation of superoxide anions was estimated by
reduction of cytochrome c. Scavenging effects of different
concentrations of all extracts on superoxide anions are shown
in Table 2. The scavenging effect of HWEL on the superoxide
anions increased with increasing concentrations of
extracts. All extracts in concentration range of 0.1–
10.0 mg/mL showed antioxidant activity, and the scavenging
rates were in the range of 21–100% (P < 0.05). In the cytochrome
c test, the IC 50 values ranged from 0.10–0.46 mg/mL,
both mung bean and adzuki bean extracts show significant
antioxidant effects (P < 0.05), and a scavenging effect on
superoxide anions.

302 Chun-Ching Lin et al.
Table 1. Inhibitory effects of different concentrations of HWEL on MDA production in mouse
liver homogenate, induced by FeCl 2 -ascorbic acid.
Concentration MDA 1 IC 50
Samples (mg/mL) nmole/mg protein Inhibition rate 2 (mg/mL)
FeCl 2 - AA + saline – 20.4 ± 0.02 –
Normal (Control) – 7.0 ± 0.05 –
FeCl 2 - AA + samples
Mung bean 0.3 12.7 ± 0.20 57.31 b 0.17
Adzuki bean 0.3 12.1 ± 0.30 61.79 a 0.15
Black bean 0.3 13.5 ± 0.36 51.20 c 0.26
Rice bean 0.3 12.8 ± 0.09 56.49 b 0.17
FeCl 2 - AA + samples
Mung bean 1.0 8.3 ± 0.13 90.07 a
Adzuki bean 1.0 9.3 ± 0.15 82.54 b
Black bean 1.0 9.8 ± 0.25 79.00 c
Rice bean 1.0 8.5 ± 0.03 89.60 a
FeCl 2 - AA + samples
Mung bean 3.0 6.8 ± 0.10 100.00 a
Adzuki bean 3.0 6.9 ± 0.23 100.00 a
Black bean 3.0 7.5 ± 0.03 96.30 bc
Rice bean 3.0 7.3 ± 0.10 97.61 b
a-tocopherol 3.0 7.2 ± 0.03 98.30 b
1 MDA data are presented as the means ± SD (n = 6).
2 The inhibitory rates within a column with different superscript letters are significantly
different at P < 0.05.
Table 2. Superoxide scavenger activities of different concentrations
of HWEL in the cytochrome c test. 1
Concentration Scavenging effect 2 IC 50
Samples (mg/mL) (%) (mg/mL)
Mung bean 0.1 42.35 b 0.14
Adzuki bean 0.1 50.00 a 0.10
Black bean 0.1 25.38 c 0.30
Rice bean 0.1 21.40 d 0.46
Mung bean 0.5 69.61 b
Adzuki bean 0.5 71.48 a
Black bean 0.5 60.70 c
Rice bean 0.5 50.25 d
Mung bean 1.0 92.84 a
Adzuki bean 1.0 92.60 a
Black bean 1.0 82.10 b
Rice bean 1.0 71.42 c
Mung bean 3.0 100.00 a
Adzuki bean 3.0 100.00 a
Black bean 3.0 92.60 b
Rice bean 3.0 78.13 c
Mung bean 5.0 100.00 a
Adzuki bean 5.0 100.00 a
Black bean 5.0 96.30 b
Rice bean 5.0 90.50 c
a-tocopherol 5.0 100.00 a
1 Data are presented as the percentage scavenging of free radicals
(n = 3).
2 Values within a column with different superscript letters are significantly
different at P < 0.05.
Anti-superoxide anion formation
HWEL had the capability of forming anti-superoxide in a
concentration-dependent manner (Table 3). All extracts at
1–15 mg/mL inhibited superoxide formation and the rates
were from 35–90%, displaying anti-superoxide formation
activity. In the xanthine-xanthine oxidase system, the formation
of superoxide was inhibited 50% (1.72–4.78 mg/mL
with extracts), with black bean extracts exhibiting potent
activity. These results indicate HWEL exhibite antisuperoxide
formation activity.
Discussion
Tissue injury caused by reactive oxygen species may include
DNA damage, lipid peroxidation (Halliwell & Gutteridge,
1984; Halliwell, 1994, 1997), protein damage (Bartold et al.,
1984), and oxidation of important enzymes (Varani et al.,
1985) in the human body.
Mouse liver homogenate was induced with FeCl 2 -
ascorbic acid for nonenzymatic lipid peroxidation. MDA is
very reactive and takes part in cross-linking DNA and proteins,
and it also damages liver cells (Kubow, 1990).
a-Tocopherol is a natural antioxidant, which functions as
a free-radical quencher in biological cells and localizes within
the phospholipid bilayer of cell membranes to protect against
biological lipid peroxidation (Hafeman & Hoekstra, 1997). It
has been confirmed that a-tocopherol decreases atherosclerosis
and delays death from myocardial infarction, presum-

Antioxidant activity of legumes 303
Table 3. Antioxidant activities of different concentrations of
HWEL in the xanthine oxidase inhibition test. 1
Concentration Inhibition rate 2 IC 50
Samples (mg/mL) (%) (mg/mL)
Mung bean 1 35.50 c 4.67
Adzuki bean 1 39.00 b 4.78
Black bean 1 45.40 a 1.72
Rice bean 1 40.97 b 3.32
Mung bean 5 55.00 c
Adzuki bean 5 49.47 d
Black bean 5 62.23 b
Rice bean 5 57.80 bc
a-tocopherol 5 70.09 a
Mung bean 10 65.00 b
Adzuki bean 10 64.70 b
Black bean 10 70.00 a
Rice bean 10 71.77 a
Mung bean 15 89.00 a
Adzuki bean 15 87.63 b
Black bean 15 88.12 ab
Rice bean 15 90.10 a
1 Data are presented as percent inhibition of superoxide formation
(n = 3).
2 Values within a column with different superscript letters are significantly
different at P < 0.05.
ably by inhibiting lipid peroxidation (Byers, 1993). Legumes
may also protect against damage to cell membranes because
they reduce the level of lipid peroxides. We can conclude from
Tables 1 and 2 that the extracts of mung bean and adzuki bean
had the strongest antioxidant action, not only in the mouse
homogenate model system but also in the cytochrome c test.
Xanthine oxidase converts hypoxanthine to xanthine and
then xanthine to uric acid in the presence of molecular
oxygen to yield superoxide anion and hydrogen peroxide, and
these radicals directly reduce ferri-cytochrome c to ferrocytochrome
c (Ho et al., 1999). Xanthine oxidase-derived
superoxide anion has been linked to post-ischaemic tissue
injury and edema as well as changes in vascular permeability
(Mc Cord & Fridovich, 1968; Hearse et al., 1986). Inhibition
of superoxide anion regeneration by the enzymatic
pathway would be beneficial in ischaemia and edema. HWEL
exhibited anti-superoxide formation activity. These observations
suggest that legumes can be prepared in beverages and
consumed in daily life. Further studies will be focused on the
isolation of active components and clarification with more
details concerning the nutritional or pharmacological effects
of legume extracts with in vitro or in vivo models.
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