Eur. J. Lipid Sci. Technol.

2008, 110, 865–878 865

Review Article
Legumes as a source of natural antioxidants

Ryszard Amarowicz1 and Ronald B. Pegg2

1
Division of Food Science, Institute of Animal Reproduction and Food Research of the Polish Academy of
Sciences, Olsztyn, Poland
2
Department of Food Science and Technology, The University of Georgia, Athens, USA

The following article summarizes the most up-to-date information available concerning endogenous
bioactives and the antioxidant activity/radical-scavenging capacity of selected leguminous seeds, and
extracts derived therefrom, as well as the impact of processing and seed germination on these bioactives.
Biologically-active compounds of interest found in leguminous seeds come from many chemical classes
and include phenolic acids as well as their derivatives, flavanols, flavan-3-ols, anthocyanins/anthocyani-
dins, condensed tannins/proanthocyanidins, tocopherols, and vitamin C. Research findings from over
100 references, many of which published only within the last 10 years, have been compiled and used in this
review.

Keywords: Antioxidant properties / Germination / Legumes / Phenolic compounds / Technological processing /

Tocopherols / Vitamin C
Received: April 28, 2008; accepted: August 27, 2008
DOI 10.1002/ejlt.200800114

1 Introduction tocopherols; vitamin C) in leguminous seeds. An important

point of consideration is the high content of phenolic anti-
Leguminous seeds are an important source of nutrient com- oxidants present in seed coats.
pounds such as protein, starch, dietary fiber, and minerals [1], This article reviews the profile and levels of phenolic
particularly in third-world countries. Incorporation of legu- compounds (i.e. total phenolics and tannins, individual flavo-
minous seeds into the human diet in developing countries can noids, and phenolic acids), tocopherols, and vitamin C in
offer protective effects against chronic diseases [2–4]. leguminous seeds. The antioxidant activity and radical-
Legumes contain a number of bioactive substances including scavenging capacity of the seeds or their extracts have been
phenolics that can diminish protein digestibility and mineral described. Finally, the influence of technological processing
bioavailability [5, 6]. On the other hand, phenolic compounds and germination on the content of natural endogenous anti-
such as flavonoids, phenolic acids, lignans, and tannins have oxidants and their antioxidative properties has been discussed.
antioxidant properties, and these are very important from
nutritional and technological points of view. Various evidences
suggest that oxidative stress is closely associated with a diverse
assortment of diseases such as cancer and cardiovascular 2 Content of total phenolics and tannins in
disease. leguminous seeds
The antioxidant capacity of legumes depends on the bio-
logical variety of the plant, and is observed over broad ranges. The total phenolic content (TPC) in leguminous seeds or
Technological processing and seed germination can impact extracts prepared from such plant materials is one of the main
the levels of natural endogenous antioxidants (e.g. phenolics, parameters dictating the potential antioxidant capacity of seeds
or the antioxidant activity of extracts therefrom. Determination
of the TPC in legumes includes an extraction step followed by a
colorimetric reaction under alkaline conditions between the
Correspondence: Ryszard Amarowicz, Division of Food Science, Insti-
tute of Animal Reproduction and Food Research of the Polish Academy
extracted phenolic constituents and Folin-Ciocalteu’s phenol
of Sciences, Tuwima Street 10, 10-747 Olsztyn, Poland. reagent. The results of the assay are reported as the quantity of
E-mail: amaro@pan.olsztyn.pl equivalents of standard compounds (i.e. typically gallic acid or
Fax: 148 89 5240124 catechin) per mass unit of raw material or extract.

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866 R. Amarowicz and R. B. Pegg Eur. J. Lipid Sci. Technol. 2008, 110, 865–878

The type of solvent used for extraction of various Table 1. Content of total phenolics in leguminous seeds and their
classes of phenolic compounds from legumes is very broad extracts.
and typical examples include water, methanol, ethanol,
methanol/water, ethanol/water, and acetone/water [7–12]. Legumes Total phenolics Unit Reference
Details pertaining to the application of different solvents Green bean 355 mg/100 g d.m.{{ [8]
for the extraction of phenolics from plant material have Pea 183
been reviewed by Naczk and Shahidi [13]. A comparative Lentil 3.76–4.17{ mg/g d.m.§§ [9]
study of phenolic profiles and antioxidant activities of 1.56–2.37{
legumes, as affected by extraction solvents, has been Green pea 1.07–1.53§ mg/g{{ [14]
reported by Xu and Chang [14]; the results of their study Yellow pea 1.13–1.67 §
showed that 50% (vol/vol) acetone extracts exhibited the Chickpea 1.41–1.67 §
highest TPC for yellow pea, green pea, and chickpea. Lentil 1,02–7.53§
Amarowicz et al. [7] reported that an acetone/water system Red kidney 1.23–5.90§
Black bean 1.28–6.89§
extracted greater quantities of phenolic compounds from
Cowpea extracts 6.45–16.36 g/100 g{{ [15]
lentil seeds compared with methanol/water or ethanol/
water systems. In the acetonic extract, thin-layer chroma- Extracts [16]
Red bean 93.6 mg/g§§
tography revealed the presence of tannins of higher mo-
Brown bean 91.4
lecular weight that were not present in ethanolic and
Black bean 44.0
methanolic extracts. Bean 18.88–25.35 mg/g§§ [17]
The TPC in legumes and extracts derived therefrom are 11.23–16.94{{
presented in Table 1. For some preparations, an absolute Milled bean samples 0.59–6.28 mg/g§§ [18]
value is given based on the reference, but in other cases, a Bean extract fractions 1.11–6.69 mg/g§§ [19]
range is provided. It is clear when examining the table that Extracts [20]
wide variations exist in the TPC, depending on the source of Faba bean 55.9 mg/g§§
the leguminous seed as well as on how it has been processed or Broad bean 23.9
extracted. Adzuki bean 89.7
Condensed tannins (i.e. proanthocyanidins) are flavan-3- Red bean 55.4
ol-based biopolymers that, at high temperature in alcohol Pea 22.6
solutions of strong mineral acids, release anthocyanidins and Red lentil 58.0
catechins as end groups. Several studies have reported on the Green lentil 67.6
antioxidant and antiradical activity of tannins [24–29]. The Extracts
most common methods used for condensed tannin analysis White bean 1.08 g/100 g§§ [21]
include the vanillin/HCl method [30], the bovine serum albu- Pea 3.48
Lentil 6.01
min (BSA) precipitation method [31], and the proanthocya-
Everlasting pea 0.97
nidin method after n-butanol/HCl hydrolysis [32]. The results
Faba bean 8.09
are generally presented as catechin equivalents per mass unit Broad bean 6.01
(i.e. the vanillin/HCl method) or as absorbance units at Bean 1.17–4.27 mg/g d.m.§§ [22]
500 nm (i.e. the vanillin/HCl method), 510 nm (i.e. the BSA Extract of vetch 22.6 mg/g§§ [23]
precipitation method) or 550 nm (i.e. the proanthocyanidin
method) per mass unit. {
Different solvents.
The condensed tannin content (CTC) in selected legu- {
Extraction with phosphate buffer.
§
minous seeds or their extracts is reported in Table 2. Lentil Extraction with 80% (vol/vol) methanol.
#
and red bean were found to be very rich sources of tannins. Extraction with 70% (vol/vol) acetone.
{{
According to Xu and Chang [14], leguminous extracts from Extraction with 50% (vol/vol) methanol.
{{
As gallic acid equivalent.
different extraction solvent systems differed significantly in §§
As catechin equivalent.
their CTC. The highest amounts of condensed tannins were
determined for lentil and black bean. The results suggested
that 80% (vol/vol) acetone was the best at extracting con- absorbance units per 1 mg of tannin fraction (A500/mg) [34].
densed tannins from the majority of the leguminous seeds. In a The findings were in the order of red bean . green lentil <
study by Madhujith et al. [16], the CTC of an extract of vetch < adzuki bean . red lentil . faba bean . field pea <
common bean hulls was several times greater than that of the broad bean. The capabilities of the tannin fraction to pre-
whole bean extract. The tannin fraction separated from legu- cipitate BSA – results reported as absorbance units at 510 nm/
minous seed extracts using Sephadex LH-20 chromatography mg (A510/mg) – were in the order of red bean < red lentil .
according to the Strumeyer and Malin method [33] was char- faba bean < vetch < field pea < broad bean . green lentil .
acterized by vanillin/HCl, and the results were expressed as adzuki bean.

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Eur. J. Lipid Sci. Technol. 2008, 110, 865–878 Antioxidants of legumes 867

Table 2. Content of condensed tannins in leguminous seeds and with colored coats are also rich in anthocyanidins [35]. The
their extracts. levels of flavonoids and phenolic acids in leguminous seeds are
reported in Tables 3 and 4.
Legumes Condensed Unit Refer- The content of total flavonoids in seeds from six legumes,
tannins ence
green pea, yellow pea, chickpea, lentil, red kidney, and black
Green pea 0.03–1.71{ mg catechin [14] bean ranged from 0.08 to 3.21 mg catechin equivalents/g [14];
Yellow pea 0.00–1.52 equivalents/g{ the highest quantity of total phenolics was determined in seeds
Chickpea 0.00–1.85 of red kidney and black bean. Flavonoids present in legumi-
Lentil 0.12–8.78 nous seeds belong to flavanols, flavan-3-ols, flavones, and
Red kidney 0.12–5.53 anthocyanidins [10, 36–41]. The majority of them, however,
Black bean 0.37–6.74 are present as glycosides in the seeds. Diaz-Batalla et al. [36]
Bean extract 0.000–0.384 mg catechin [16] also detected isoflavones in germinated beans.
Bean hull extract 0.000–2.677 equivalents/g{ In the study of Sosulski and Dabrowski [42], the phenolic
Bean extract 0–717 A550/g§
constituents in defatted flours and hulls of ten leguminous
Bean hull extract 0–3830
species, mung bean, smooth field pea, yellow lentil, small faba
Extracts bean, pigeon pea, navy bean, white lupine, baby lima bean,
Faba bean 0.545 A550/mg§ [20]
chickpea, and cow pea, were fractionated into free acids, sol-
Broad bean 0.156
uble esters, and residue compounds. The flours contained
Adzuki bean 1.754
Red bean 1.753 only soluble esters; hydrolysis of these revealed the presence of
Pea 0.203 trans-ferulic, trans-p-coumaric, and syringic acids in nearly all
Red lentil 0.934 of the species examined. The lowest amount of phenolic acids
Green lentil 0.829 was found in mung bean, field bean, lentil, faba bean, and
Bean 0.22–1.28 mg catechin [22] pigeon pea, with 2–3 mg of phenolic acids per 100 g of flour.
equivalents/g{ Navy bean, lupine, lima bean, and cowpea were characterized
Vetch 71 A500/g{ [23] as possessing the highest level of phenolic acids. The hulls
Tannin fractions separated contained p-hydroxybenzoic, protocatechuic, syringic, gallic,
from extracts trans-p-coumaric, and trans-ferulic acids in the soluble ester
Red bean 0.311 A500/g{ [34] fraction. Madhujith et al. [12] reported vanillic, caffeic, p-
Adzuki bean 0.256 coumaric, ferulic, and sinapic acids as the main phenolic acids
Red lentil 0.205 identified in bean hull extracts.
Green lentil 0.285
The content of procyanidin B2, B3, and procyanidin tet-
Broad bean 0.066
Faba bean 0.195
ramer in lentils ranged from 0.1 to 0.5 mg/100 g dry weight
Field pea 0.072 (d.w.) [39]. In an extract of adzuki bean, the contents of pro-
Vetch 0.269 cyanidin dimers and trimers ranged from 15.9 to 213 mg/g
[40]. Procyanidin B2, C1, and C2 were identified in red,
Tannin fractions separated
from extracts brown, and black bean hull extracts [12]. The most abundant
Red bean 0.030 A510/g [34] proanthocyanidins in the lentil seed coat were polymers with a
Adzuki bean 0.006 mean degree of polymerization (mDP) of 7–9, followed by
Red lentil 0.026 oligomers with an mDP of 4–5 [43].
Green lentil 0.09
Broad bean 0.011
Faba bean 0.015 4 Content of tocopherols and vitamin C in
Field pea 0.012 leguminous seeds
Vetch 0.014

{
Table 5 summarizes the content of tocopherols present in
Results were obtained using several solvent systems.
{ selected leguminous seeds. Although no tocotrienols have
Results were obtained using the vanillin/HCl method.
§
Results were obtained using the proanthocyanidin method. been detected, all four tocopherol isomers are present. How-
#
Results were obtained using the BSA precipitation method. ever, the levels of these isomers vary quite markedly, with g-
tocopherol being the predominant one. The greatest g-toco-
pherol levels were reported in lentils and peas. Recent studies
suggest that g-tocopherol does not get the respect it deserves
3 Phenolic composition of leguminous seeds as a nutrient. g-Tocopherol may have unique functions in
detoxifying nitrogen dioxide and other reactive nitrogen spe-
The dominant phenolic compounds present in leguminous cies. The effect of a- and g-tocopherol supplementation on
seeds are flavonoids, phenolic acids, and procyanidins. Seeds platelet aggregation and thrombosis in rats revealed that

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868 R. Amarowicz and R. B. Pegg Eur. J. Lipid Sci. Technol. 2008, 110, 865–878

Table 3. Content of flavonoids in leguminous seeds and their extracts.

Legumes Compound Content Unit Reference

Green pea Total flavonoids 0.08–0.39 mg catechin equivalent/g [14]

Yellow pea 0.18–0.32
Chickpea 0.18–3.16
Lentil 0.72–2.21
Red kidney 0.85–2.93
Black bean 1.19–3.21
Bean Total flavonoids 0.22–1.43 mg catechin equivalent/g [22]
Pea seed cotyledon (ZP-849 and Fidelia (–)-Epigallocatechin nd; 1.61 mg/g d.m. [10]
variety) Kaempferol 3-rutinoside-7-rhamnoside 1.12; nd
Apigenin 8-C-glucoside 0.80; 1.31
Quercetin 3-O-galactoside
Pea seed coat (ZP-849 and Fidelia variety) (–)-Epigallocatechin 78.82; 9.35 mg/g d.m. [10]
(1)-Catechin nd; 8.08
(–)-Epicatechin 7.62; nd
Luteolin glycoside 20.40; 50.65
Apigenin 8-C-glucoside 74.35; 149.9
Quercetin 3-O-galactoside nd; 0.16
Quercetin 3-O-rhamnoside 12.08; nd
Kidney bean coat Cyanidin 3,5-diglucoside 0.000–0.043 mg/g [35]
Delphinidin 3-glucoside 0.000–0.182
Cyanidin 3-glucoside 0.000–0.125
Petrunidin 3-glucoside 0.000–0.167
Pelargonidin 3-glucoside 0.000–0.588
Bean Quercetin{ 6.9–23.5 mg/g d.m. [36]
Kaempferol{ 13.8–209.4
Germinated bean Quercetin{ 13.3–50.5 mg/g d.m. [36]
Kaempferol{ 0.0–39.6
Daidzein 8.2–43.6
Genistein 2.6–9.7
Coumestrol 2.4–11.7
Cowpea Quercetin 3-O-galactoside 3.64 mg/g [37]
Quercetin 3-O-glucoside 11.45
Quercetin diglycoside 1.80
Myricetin 3-O-glucoside 9.64
Pea extract Quercetin 0.14 mg/g [38]
Kaempferol 0.51
Lentil (1)-Catechin 0.1–0.3 mg/100 g d.m. [39]
Adzuki bean extract Epicatechin 25.7 mg/g [40]
Epigallocatechin gallate 0.14
Epicatechin glucoside 159.0
Catechin glucoside 688.0
Quercetin 36.2
Quercetin rutinoside 38.2
Quercetin galactoside 46.9
Quercetin arabinoglucoside 181.0
Lentil seed coat Catechins 919–1633 mg/g [41]
Glycosides of flavones 33.1–186
Glycosides of flavonols 9.6–241

nd, Not detected.

{
After acidic hydrolysis.
{
Different solvents.

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Eur. J. Lipid Sci. Technol. 2008, 110, 865–878 Antioxidants of legumes 869

Table 4. Content of phenolic acids in leguminous seeds and their extracts.

Legumes Compound Content Unit Reference

Pea seed cotyledon (ZP-849 and Fidelia Protocatechuic 2.77; 19.82 mg/g d.m. [10]
variety) Glycoside of protocatechuic 42.28; 14.97
p-Hydroxybenzoic 4.69; 16.62
Vanillic nd; 3.18
trans-p-Coumaroylmalic 0.71; 0.66
cis-p-Coumaric nd; 0.46
Syringic nd; 0.24
trans-Ferulic nd; 0.99
Pea seed coat (ZP-849 and Fidelia variety) Gallic 14.21; 70.91 mg/g d.m. [10]
Protocatechuic 50.12; 76.99
p-Hydroxybenzoylmalic nd; 30.19
p-Hydroxybenzoic 23.27; 35.25
Vanillic nd; 11.95
trans-p-Coumaroylmalic 13.47; 28.95
trans-p-Coumaric nd; 1.48
trans-Feruloylmalic nd; 19.06
trans-Ferulic nd; 4.49
Germinated bean p-Hydroxybenzoic 1.5–4.1 mg/g d.m. [36]
Vanillic 7.7–46.3
p-Coumaric nd–15.5
Ferulic 16.7–41.9
Cowpea Gallic 0.16 mg/g [37]
Protocatechuic 1.21
trans-Feruloylaldaric 4.01
trans-p-Coumaroyaldaric 3.66
p-Hydroxybenzoic 4.49
Vanillic 2.51
trans-p-Coumaric 0.86
trans-Ferulic 1.60
cis-Ferulic 1.24
Pea extract Vanillic{ 0.07 mg/g [38]
Caffeic 0.02
p-Coumaric 0.06
Ferulic acid 0.32
Sinapic 0.07
Lentil Protocatechuic 49.9–52.3 mg/100 g d.w. [39]
p-Hydroxybenzoic 93.6–100
Vanillic 13.3–15.3
trans-p-Coumaric 322–342
trans-Ferulic 20.9–25.7
Bean Protocatechuic 32.8–41.4 mg/100 g d.w. [39]
p-Hydroxybenzoic 32.3–36.1
trans-Ferulic 342–366
Adzuki bean extract Protocatechuic 67.6 mg/g [40]
trans-p-Coumaric 31.3
trans-p-Coumaroylmalic 4.57
Lentil cotyledons Hydroxybenzoic acids 1.8–2.2 mg/g [41]
Free hydroxycinnamic acids 3.2–5.7
Combined hydroxycinnamic acids 1.4–13.5

nd, Not detected.

{
After basic hydrolysis.

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870 R. Amarowicz and R. B. Pegg Eur. J. Lipid Sci. Technol. 2008, 110, 865–878

g-tocopherol leads to a greater decrease in platelet aggregation 5 Antioxidant activity of leguminous seeds or
and delay of arterial thrombogenesis than a-tocopherol sup- their extracts
plementation. Additionally, there has been evidence to suggest
that g-tocopherol may be protective against cardiovascular The antioxidant and antiradical activities of leguminous
disease because plasma g-tocopherol levels were inversely extracts were investigated using methods such as storage
associated with increased morbidity and mortality due to car- studies of oils [16, 58], liposomes [59, 60], a b-carotene-
diovascular disease in population studies. linoleate model system [11, 16, 18, 19, 28, 29, 38, 40, 61–67],
Leguminous seeds are not a rich source of vitamin C [52], an electron spin resonance (ESR) spin-trapping technique
but this vitamin’s contribution to the antioxidant capacity of [68], enhanced chemiluminescence and photo-
seeds, especially for the part generated by hydrophilic com- chemiluminescence [21, 69], scavenging of 2,20 -azobis(2,4-
pounds, is high. In foods of plant origin, vitamin C is deter- dimethylvaleronitrile) (AMVN) radicals [61, 70], 2,20 -azobis-
mined as ascorbic and dehydroascorbic acid. The level of (2-amidopropane) (ABAP) radicals [71], 2,20 -diphenyl-1-
vitamin C in leguminous seeds is summarized in Table 6. picrylhydrazyl (DPPH) radicals [14, 22, 23, 38, 40, 43, 65,
The content of ascorbic acid in peas ranged from 0.40 to 72–74], 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic
1.48 mmol/g [50]; it was positively correlated with the total acid) (ABTS) cation radicals [20, 23, 38, 51, 75], superoxide
antioxidant capacity of the water-soluble compounds anion radicals [74–76], peroxyl radicals [76], reducing power
(r = 0.72; p ,0.001). In the study of Moriyama and Oba [56], [23, 38, 40, 65], LDL cholesterol oxidation [76, 77], Fe21-
the average value of total vitamin C per 100 g of dry weight in chelating capacity [76, 78], the ferric-reducing antioxidant
dehydrated samples varied from 0.24 (kidney beans) to power (FRAP) assay [15, 56, 73, 79, 80], and the oxygen
4.14 mg (green peas). In green pea, the majority of vitamin C radical absorbance capacity (ORAC) assay [73, 80, 81]. Some
was present as dehydroascorbic acid. of the findings are reported in Table 7.

Table 5. Content of tocopherols in leguminous seeds.

Legumes a-T b-T g-T d-T Unit Reference

Lentil 3.84–8.69 1.94–3.81 91.11–104.68 2.01–2.74 mg/g d.m. [9]

Green pea{ 0.02 mg/100 g edible wt [44]
Green pea{ 0.02
Butter beans 0.7 4.7§ mg/100 g [45]
Chickpea 6.9 5.5§
Kidney bean 1.2 2.6§
Lentil 1.6 4.5§
Pea 10.4 5.7§
Pea 90.4–97.3 mg/kg [46]
Broad bean 16.6 53.2 – mg/kg [47]
Bean – 34.1 6.3
Flat pea 7.8 87.2 –
Field pea 7.2 79.2 6.2
Garden pea 7.1 85.3 –
Horse bean 15.3 50.3 5.4
Kidney bean – 54.5 7.2
Lentil 10.1 66.5 –
Bayo bean – 2.64 mg/100 g wet weight [48]
Black bean – 0.90
Pinto bean 0.20 2.6
Garbanzo bean 2.81 7.29
Faba bean 0.95 5.22
Lentil 1.1 4.66
Split pea 0.12 4.65
Grass pea 1.78 0.21 53.54 mg/g [49]
Cowpea – – 0.43 1.83 mg/100 g d.m. [50]
Pigeon pea 1.6 0.06 9.31 0.27 mg/100 g d.m. [51]
{
Blanched, frozen.
{ Blanched, canned.
§
b 1 g.
#
a 1 g 1 d.

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Eur. J. Lipid Sci. Technol. 2008, 110, 865–878 Antioxidants of legumes 871

Table 6. Content of vitamin C in leguminous seeds. liver microsomal system and the scavenging effect of hydroxyl
radicals and superoxide anion radicals were influenced by the
Legumes Content Unit Refer- chemical composition of the anthocyanins [83]. The anti-
ence
oxidative activity of aglycones increased in the order pelargo-
Mung bean 6.27 6 1.09{; mg/100 g f.w. [52] nidin , cyanidin , delphinidin. The findings suggested that
0.23 6 0.05 as the number of hydroxyl substituents on the B ring
Adzuki bean 2.59 6 0.28{; increased, a greater activity was achieved by the glucosides,
0.02 6 0.01 whereas for the aglycones, an increase in hydroxyl sub-
Black gram 0.45 6 0.04{; stituents resulted in weaker activity.
0.00 The activity of water-soluble antioxidants in the extracts of
Kintoki bean 0.79 6 0.12{; six legumes investigated using photochemiluminescence
0.06 6 0.00§ exhibited the following trend: faba bean . lentil . broad bean
Tora bean 0.05 6 0.01{;
. pea . everlasting pea . white bean [21]. The strongest
0.44 6 0.02
antioxidant properties, due to lipid-soluble antioxidants
Broad bean 0.21–1.94{;
0.00–0.06
determined by the photochemiluminescence method, were
Green pea 0.00–1.74{; from extracts of lentil and pea, while less activity was noted for
0.32–6.24 extracts of everlasting pea and faba bean. In the same study,
Pea 32 6 1 mg/100 g f.w. [53] results of antioxidant activity of leguminous seed extracts
Pea{ 10.0{ mg/100 g f.w. [54] evaluated by the enhanced chemiluminescence method were
Grass pea [55] in the order of lentil . faba bean . pea . broad bean .
Derek cultivar 26.8{; 36.3§ mmol/g everlasting pea . white bean.
Krab cultivar 25.7{; 26.7§ The results of a b-carotene-linoleate test indicated that the
Pea 0.40–1.48{ mg/100 g d.w. [56] extracts from pea, faba bean, lentil, everlasting pea, and broad
Bean 12.0§ mg/100 f.w. [57]
bean seeds had similar antioxidative activity, while the extract
{
from white bean seeds was clearly less active. After 60 and
Blanched. 120 min of incubation at 50 7C, about 75–80% and 61–66% of
{
Ascorbic acid.
§
Ascorbic 1 dehydroascorbic acid.
the b-carotene remained unoxidized, respectively, for systems
#
Dehydroascorbic acid. treated with the more active extracts. For white beans, the re-
spective values were 58.8 and 32.4% [62]. The antioxidant
activity of fractions separated from leguminous seed extracts
Strong antioxidant activity evaluated with a linoleic acid by Sephadex LH-20 column chromatography and then eval-
system was found in the hydrophilic phenolic extract of pea uated using the b-carotene-linoleate model system was in the
bean [59]. The same extract had a synergistic effect with a- order of everlasting pea . faba bean . broad bean [63].
tocopherol in a linoleic acid and a liposome system. Navy bean The results of Madhujith et al. [16] demonstrated that
hull extracts proved to offer superior antioxidant activity than colored beans possessed superior antioxidant activity com-
that of a mixture of butylated hydroxyanisole (BHA) and pared to white beans. Evaluation of antioxidant activity in an
butylated hydroxytoluene (BHT) when used at similar con- emulsion system revealed that red, brown, and black whole
centrations in storage studies (26 and 37 7C, 9 months) of soy bean extracts were capable of inhibiting oxidation of b-caro-
and sunflower oils [58]. tene (ca. 33–52%) as compared to the control. In a corn oil
Among cool-season leguminous classes, lentils possessed model system, red, brown, and black bean extracts inhibited
the highest concentrations of phenolic compounds and anti- the formation of conjugated dienes (20–28%), 2-thiobarbi-
oxidant activities. Colored (i.e. anthocyanin/anthocyanidin turic acid-reactive substances (44–52%), and hexanal (68–
rich) common beans exhibited higher FRAP, DPPH., and 84%) when used at the 100-ppm level as catechin equivalents.
ORAC values than those of yellow peas, green peas, and The hydrogen peroxide-scavenging capacity of different
chickpeas. Antioxidant activities (FRAP, DPPH., and ORAC) beans (i.e. white kidney, red pinto, Swedish brown, black kid-
were strongly correlated (r = 0.96, 0.94, and 0.89, respec- ney) ranged from 58 to 67% at 50 ppm and from 65 to 76% at
tively, p ,0.01) with total phenolic content [73]. 100 ppm [77]. All extracts employed retarded human low-
Anthocyanin pigments isolated from Phaseolus vulgaris density lipoprotein cholesterol oxidation by 61.4–99.9% at
seed coats (i.e. pelargonidin 3-O-b-D-glucoside, cyanidin 3- levels of 2–50 ppm of catechin equivalents.
O-b-D-glucoside, and delphinidin 3-O-b-D-glucoside) and The results of Fernandez-Orozco et al. [9] showed a very
their counterpart aglycones (i.e. pelargonidin chloride, cyani- high molar percentage contribution of phenolic compounds
din chloride, and delphinidin chloride) exhibited strong anti- and a low contribution of tocopherols, glutathione, soluble
oxidant activities in a liposomal system and reduced the for- proteins, and ascorbate (only in germinated seeds) to the total
mation of malondialdehyde by UV radiation. The extent of trolox equivalent antioxidant capacity (TEAC) and peroxyl
antioxidant activity afforded by anthocyanin pigments in a rat radical-trapping capacity (PRTC), calculated as a sum of data

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872 R. Amarowicz and R. B. Pegg Eur. J. Lipid Sci. Technol. 2008, 110, 865–878

Table 7. Antioxidant capacity/activity of leguminous seeds or their extracts.

Legumes Capacity/activity Unit Method Reference

Cowpea extracts 7.7–69.7 Scavenging effect (%) – Superoxide radical-scavenging [15]

concentration: 200–400 mg activity
285–662 mmol trolox/kg ABTS.1
705–1922 EC50 (mg/g DPPH.) DPPH.
487–1560 mg/mmol Fe(II) FRAP
Beans extracts 28.59–51.51 Unoxidized b-carotene (%) b-Carotene bleaching [16]
Milled bean samples 16.6–31.9 AA (%) b-Carotene bleaching [18]
27.7–32.6 ARA (%) DPPH.
Germinated [39]
peas 6.7–14.5 IC50 (mg of legume flours) DPPH.
beans 7.5–22.6
lentils 7.2–10.9
Pea Water-soluble phenolics: mmol trolox/g FRAP [56]
0.61 6 0.23
(0.21–1.29); n = 108
Water insoluble phenolics:
0.23 6 0.08
(0.05–0.46); n = 105
Red, black, white bean 89.5–100.0 Retention of supercoiled DNA (%) RI-DNA-D [74]
Yellow pea 1.58 mmol trolox/g LDL-CD [77]
Green pea 1.50
Chickpea 1.23
Black bean 29.53
Lentil 28.37
Red kidney 22.55
Pinto bean 14.98
Pea 30–72 mg AAE/100 g d.m. FRAP [79]
Green bean 90–150
Broad bean 1.86 mmol/100 g FRAP [80]
Pinto bean 1.14
Black-eyed bean 1.08
Lentil 0.49
Kidney bean 0.38
Mung bean 0.35
Chickpea 0.23
Garden pea 0.12
Green pea 9.57 mmol trolox/g ORAC [81]
Yellow pea 12.06
Chickpea 18.66
Lentil 94.90
Lima bean & Jack bean 35.5 AA (%) Linoleic acid oxidation [82]
extracts 39.4

AA, antioxidant activity; AAE, ascorbic acid equivalents; ARA, antiradical activity; RI-DNA-D, radical-induced DNA damage; LDL-CD, low-
density lipoprotein conjugated dienes; FRAP, ferric-reducing antioxidant power; ORAC, oxygen radical absorbance capacity; DPPH., 2,20 -
diphenyl-1-picrylhydrazyl radical; ABTS.1, 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) cation free radical; IC50, inhibitory con-
centration at 50%; EC50, effective concentration at 50%.

provided for phosphate-buffered and 80% (vol/vol) methanolic According to Lee et al. [84], aroma extracts isolated
extracts of raw and processed lentil seeds. Beninger and Hos- from mung bean, kidney bean, and adzuki bean inhibited
field [60] concluded their investigation by stating that pure fla- oxidation of aldehydes to carboxylic acids and oxidation
vonoids such as anthocyanidins, quercetin glycosides, and of cod liver oil. The antioxidant activity of mung
condensed tannins, which are present in the seed coats of com- bean aroma extract was comparable with that of a-toco-
mon beans, have significant antioxidant activity relative to BHT. pherol.

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Eur. J. Lipid Sci. Technol. 2008, 110, 865–878 Antioxidants of legumes 873

6 Change in the content of natural mentation. The marked increase in quercetin levels was
antioxidants in leguminous seeds during accompanied by a decrease in quercetin 3-O-glucoside and
technological processing and germination quercetin 3-O-galactoside. After spontaneous fermentation,
an increase in the free phenolic acids was also observed. Yet,
Sosulski and Dabrowski [42] noted that the dehulling process after fermentation with Lactobacillus plantarum, a decrease in
substantially reduced the phenolic acid composition of flour trans-p-coumaric and cis-ferulic acids was noted. The bacteria
from leguminous seeds for pigeon pea, faba bean, mung bean, strains employed in the experiment were probably able to
and lentil, but had little effect on the content of phenolic acids impart phenolic acid decarboxylase activity, which is in
of flour from field pea, navy bean, and chickpea. accordance with the findings of Cavin et al. [90].
During the soaking period, the content of phenolic acids in The germination process of leguminous seeds modified
leguminous seeds (i.e. beans, peas, and lentils) decreased; this their phenolic composition. The metabolic changes seem to
was observed for bean, pea, and lentil by López-Amorós et al. be affected by the period of germination and by light [39]. The
[39] and for pea by Bishnoi and Khetarpaul [85]. Soaking of content of phenolic acids (i.e. p-hydroxybenzoic, vanillic, p-
lentils removed procyanidin B2, B3 and C1, and the procyani- coumaric, and ferulic acid) decreased after germination of
din tetramer from seeds [39]. Washing (i.e. three times with pea, bean, and lentil seeds. The presence of vanillic and p-
sterile distilled water under aseptic conditions) and drying (at hydroxybenzoic acids was detected only in the germinated
55 7C for 24 h) modified the phenolic acid composition of seeds; this could have been caused by the degradation of the
cowpea seeds, but only slightly [37]. seed lignin by way of enzymatic oxidation [91]. In the case of
The phenolic content in common beans after pressure beans, the presence of flavonol glycosides (i.e. quercetin-3-
cooking (i.e. 121 7C; 103.421 kPa; 5, 7 or 60 min) was rutinoside, quercetin-3-rhamnoside, kaempferol-3-rutino-
reduced drastically; in the case of seed coats, the reduction was side, and kaempferol-3-glucoside) was detected only in the
by 90% [86]. During the longer cooking times, phenols from germinated seeds. The highest concentrations of flavonols
seed coats diffused into the cooking water and from there to were observed in the seeds germinated with light [39]. The
the cotyledons. The content of phenolic compounds extracted procyanidins B2, C1, and the procyanidin tetramer were only
from cooked (up to 30 min) lentil seeds using phosphate buf- observed in raw lentil seeds. The content of quercetin in ger-
fer increased from 52 to 72%. When 80% (vol/vol) methanol minated bean seeds was higher than the respective values
was used for extraction, the content of total phenolics in the found in the raw starting material. For kaempferol, the
same material after cooking was 134 to 154% of the quantities reversed effect of germination was observed [36]. In germi-
determined in the raw seeds [9]. After cooking (i.e. boiling for nated bean seeds, the presence of daizein, genistein, and cou-
5 min or microwave cooking for 1 min or steaming for mesterol was observed. In raw and cooked seeds, isoflavones
7.5 min), the TPC of peas was significantly (p ,0.05) were completely absent.
reduced. In the case of green beans, the TPC increased after According to Dueñas et al. [72], hydroxycinnamic com-
cooking, and this is likely due to higher extractability of phe- pounds and proanthocyanidins significantly decreased after
nolic compounds from the processed material [8]. Barroga et enzymatic treatment with a-galactosidase, phytase, visco-
al. [87] found that boiling and steam cooking reduced the zyme, and tannase. However, quercetin 3-O-rutinoside and
quantity of phenolic compounds in mung bean (Vigna luteolin increased and reached their highest concentration
radiata) by 73%. The diffusion of phenolics into the cotyle- after treatment with tannase. Catechins, procyanidins and
dons was also noted. The reduction in the content of quercetin prodelphinidins experienced a decrease compared to the raw
and kaempferol in common bean seeds after processing (i.e. lentil by all enzymatic treatments.
the seeds were autoclaved for 20 min at a 1 : 3 beans/deionized The cooking process and germination can also modify the
water ratio) ranged from 12 to 65% and from 5 to 71%, content of tocopherols in leguminous seeds. Extrusion
respectively [36]. reduced the content of tocopherols in grass pea (Lathyrus
Fermentation of leguminous seeds can modify their phe- sativus L.) [47]. In particular, g-tocopherol decreased signifi-
nolic composition. In spontaneously fermented lentils, p- cantly with an increased moisture content of the seeds before
hydroxybenzoic and protocatechuic acids as well as (1)-cate- extrusion. Cooking of Mexican leguminous seeds (i.e. bayo
chin increased whereas hydroxycinnamic acids and procyani- bean, black bean, pinto bean, garbanzo bean, faba bean, lentil,
din dimers decreased [88]. An increase in some phenolic and split pea) resulted in a loss of g-tocopherol ranging from
constituents was also observed during the fermentation of 10 (black bean) to 17% (garbanzo bean) [48]. After cooking
beans [89]. Fermentation of cowpeas (Vigna sinensis L.) with the lentil seeds of different cultivars (i.e. 30 min in a flask with
natural microflora and with Lactobacillus plantarum modified a reflux condenser), tocopherol analysis of the seeds revealed
the content of phenolic compounds, but the modification was 12.5–28.7% a-tocopherol, 9.3–55.5% b-tocopherol, 18.3–
different in each case [37]. This process gave rise to tyrosol 65.5% g-tocopherol, and 61.7–95.2% d-tocopherol present in
and quercetin, which were not detected in the raw cowpea the raw seeds [9]. The percentage of a-, b-, g-, and d-toco-
flour. The levels of these two compounds were more abundant pherol in germinated lentil seeds in relation to the raw seeds
after inoculation of the cowpeas than by spontaneous fer- ranged from 101.6 to 148.9, 57.5 to 66.3, 44.6 to 70.7, and

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

874 R. Amarowicz and R. B. Pegg Eur. J. Lipid Sci. Technol. 2008, 110, 865–878

53.3 to 77.3, respectively. The preparation of cowpea seeds parison with the content determined before culinary proces-
for fermentation caused a decrease in g- and d-tocopherol (i.e. sing. Hajare et al. [97] observed that treatment with irradiation
28 and 26%, respectively) [50]. Fermentation with Lactoba- (1 and 2 kGy) and storage did not have any significant effect
cillus plantarum and with natural microorganisms present in on the vitamin C content of the control as well as irradiated
the flour brought about sharp decreases in the g- and d-toco- sprout samples of pea and garden pea stored at 4 and 8 7C.
pherol contents. In fermented flour, b-tocopherol was detec- Treatment of mung bean sprouts with a dose of 1.5 and 2 kGy
ted, but was absent from the raw starting material. Natural significantly reduced the total vitamin C content [98]. For
fermentation of pigeon pea decreased the content of g-toco- mung bean, matki bean, chana pea, and vatana pea, the con-
pherols by one-third, whereas the contents of a-, b-, and d- tent of vitamin C in the sprouts of seeds treated with irradia-
tocopherols were not reduced [51]. tion up to 2.0 kGy remained unaffected [99].
The quantity of total ascorbic acid in peas stored at 4 7C The increase in total ascorbate (i.e. reduced ascorbate 1
rapidly decreased within 14 days (i.e. 39% of the original dehydroascorbate) was observed both in embryo axes and
amount was lost). After 7 days of storage at 20 7C, the amount cotyledons during germination. After 24 h, the amount of
of total ascorbic acid decreased by 72%. A similar decrease in total ascorbate increased by ,2.9 and ,1.5 times in embryo
total ascorbic acid content was observed for green beans [79]. axes and cotyledons, respectively, as compared to dry seeds
Peas stored at –18 7C showed losses between 12 and 32% of [100]. In the study of Fernandez-Orozco [9], ascorbic acid
their original total ascorbic acid content [92]. Approximately was detected only in germinated lentil seeds in the range of
40% of the original ascorbic acid content in green beans was 0.435–0.715 mg/g dry mass (d.m.). An overall significant lin-
lost after 3 days of storage at 4 7C [93]. Serpen et al. [94] ear relation was determined between vitamin C biosynthesis
studied the time-dependent changes during frozen storage of and germination time up to 120 h at ambient room tempera-
peas; the changes in both ascorbic acid and dehydroascorbic ture in chickpea [101].
acid were strongly correlated with the kinetic model described
here. Blanching resulted in a 19% reduction in the k1 value (i.e.
the ascorbic acid degradation rate constant) as compared to 7 Change in the antioxidant activity of
unblanched peas (0.227/month 6 5.43610–3/month). The leguminous seeds during technological
regeneration rate constant of ascorbic acid (k2) increased ap- processing and germination
proximately 26-fold for blanched peas when compared to
unblanched peas (0.0114/month 6 1.04610–3/month). The Although the level of phenols was low for cooked beans, their
rate constant (k3) for the conversion of dehydroascorbic acid antiradical activity against DPPH. was similar to that meas-
to 2,3-diketogluconic acid in blanched peas decreased ,31- ured for crude seed coats and higher than that noted for crude
fold by the blanching treatment. The content of vitamin C in cotyledons [17]. The total antioxidant capacity (TAC) of
green bean decreased during blanching according to a first- green beans measured using the DPPH.-scavenging method
order reaction [95]. increased during the cooking procedure, whereas the TAC of
Blanching decreased the vitamin C content in broad bean peas remained the same as for fresh samples according to each
by 14–43%. Freezing of blanched seeds and a 6-month storage type of cooking method [8]. The process of soaking, boiling,
of frozen goods decreased this content by ,24–56%. Frozen and steaming of green pea, yellow pea, chickpea, and lentil
whole seeds contained 9.3–13.8 mg vitamin C in 100 g after caused significant decreases in DPPH.-scavenging activity in
cooking (compared with fresh seeds, the losses reached 56– all legumes as compared to the original unprocessed samples
73%), whereas appertized canned seeds contained 8.2– [82]. All soaking and atmospheric boiling treatments caused a
11.7 mg in 100 g (compared with fresh seeds, the losses significant decrease in ORAC values. However, pressure boil-
reached 63–73%) [96]. Almost 30% of the vitamin C was ing and steaming increased the ORAC values of leguminous
destroyed during blanching of grass pea seeds [55]. At the end seeds.
of the food service cycle, the ascorbic acid losses in peas pre- The treatment of lentil flour with enzymes (i.e. viscozyme,
pared in a hospital cook-chill plated system were 42% [54]. By a-galactosidase, and tannase) generated an increase in the
soaking for 16 h in the dark at 20 7C, the total vitamin C con- antioxidant activity as measured by the DPPH. test when
tent of black and mung beans increased by 3.1- and 4.5-fold, compared to raw lentils [72]
respectively. However, the varieties of green peas significantly Spontaneous and inoculated fermentation of cowpea pro-
lost their total vitamin C during the same soaking treatment. duced an increase in the antioxidant activity of phenolic com-
Boiling of dehydrated seeds of adzuki beans, broad beans, and pounds present in seeds as assayed by the DPPH. test [37]. An
green peas resulted in ,70–100% loss of total vitamin C [52]. increase in antioxidant activity of phenolics from faba bean
The cooking of grass seeds reduced the content of vitamin C and oat tempeh was reported by Berghofer et al. [102]. As
by 10 and 11%, depending on the cultivar, while the share of compared to commercial antioxidants, red bean extract fer-
ascorbic acid fell to 45% in the amount of vitamin C [55]. mented by Bacillus subtilis and Aspergillus oryzae showed less
Vanderslice et al. [57] found 45% reduction of vitamin C of a scavenging effect on DPPH. and a weaker reducing power
content after cooking frozen seeds of common pea in com- compared to that of a-tocopherol and BHT, but better Fe21-

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Eur. J. Lipid Sci. Technol. 2008, 110, 865–878 Antioxidants of legumes 875

chelating capacity [103, 104]. After pigeon pea fermentation, capacity of raw and processed lentil seeds. Nahrung/Food 2003,
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