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Critical Reviews in Plant Sciences
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The Role of Grain Legumes in the Prevention of
Hypercholesterolemia and Hypertension
Anna Arnoldia, Chiara Zanonia, Carmen Lammia & Giovanna Boschina
a Department of Pharmaceutical Sciences, University of Milan, I-20133 Milano, Italy
Published online: 24 Oct 2014.
To cite this article: Anna Arnoldi, Chiara Zanoni, Carmen Lammi & Giovanna Boschin (2015) The Role of Grain Legumes
in the Prevention of Hypercholesterolemia and Hypertension, Critical Reviews in Plant Sciences, 34:1-3, 144-168, DOI:
10.1080/07352689.2014.897908
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Critical Reviews in Plant Sciences, 34:144–168, 2015
Copyright C
Taylor & Francis Group, LLC
ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352689.2014.897908
The Role of Grain Legumes in the Prevention of
Hypercholesterolemia and Hypertension
Anna Arnoldi, Chiara Zanoni, Carmen Lammi, and Giovanna Boschin
Department of Pharmaceutical Sciences, University of Milan, I-20133 Milano, Italy
Table of Contents
I. INTRODUCTION ...............................................................................................................................................................................................145
II. THE “BIG FOUR” FUNCTIONAL INGREDIENTS FOR DYSLIPIDEMIA PREVENTION .................................... 145
A. Soybean Protein ............................................................................................................................................................................................146
B. Soluble Fiber ..................................................................................................................................................................................................147
C. Phytosterols ....................................................................................................................................................................................................147
D. Omega-3 Fatty Acids ..................................................................................................................................................................................147
III. NUTRITIONAL COMPONENTS OF GRAIN LEGUMES RELEVANT IN DYSLIPIDEMIA PREVENTION 148
A. Proteins ............................................................................................................................................................................................................148
B. Carbohydrates ................................................................................................................................................................................................149
C. Lipids ................................................................................................................................................................................................................149
D. Final Considerations ...................................................................................................................................................................................150
IV. HYPOCHOLESTEROLEMIC ACTIVITY OF GRAIN LEGUMES ........................................................................................150
A. Animal Studies .............................................................................................................................................................................................. 150
1. Chickpea, cowpea, common bean, faba bean, lentil and pea ...................................................................................................150
2. Lupin ............................................................................................................................................................................................................152
B. Human Studies ..............................................................................................................................................................................................153
C. Mechanism of the Hypocholesterolemic Activity ............................................................................................................................157
1. Soybean .......................................................................................................................................................................................................158
2. Pea .................................................................................................................................................................................................................159
3. Lupin ............................................................................................................................................................................................................159
4. Final considerations ................................................................................................................................................................................159
V. HYPOTENSIVE ACTIVITY OF GRAIN LEGUMES ......................................................................................................................159
A. Animal and Clinical Studies .....................................................................................................................................................................160
1. Mixed grain legumes ..............................................................................................................................................................................160
2. Lupin ............................................................................................................................................................................................................160
B. In Vitro Studies on ACE-Inhibitory Peptides .....................................................................................................................................160
1. Soybean .......................................................................................................................................................................................................161
2. Peanut, chickpea, and lentils ................................................................................................................................................................161
3. Common bean, Lima bean, cowpea, and mung bean .................................................................................................................162
4. Pea .................................................................................................................................................................................................................162
5. Lupin ............................................................................................................................................................................................................163
6. Final considerations ................................................................................................................................................................................163
Address correspondence to Anna Arnoldi, Department of Pharmaceutical Sciences, University of Milan, I-20133 Milano, Italy. E-mail:
anna.arnoldi@unimi.it
144
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GRAIN LEGUMES 145
VI. CONCLUSIONS ...................................................................................................................................................................................................163
ACKNOWLEDGMENT ..................................................................................................................................................................................................163
FUNDING ..............................................................................................................................................................................................................................163
REFERENCES ....................................................................................................................................................................................................................163
The seeds of the plants of the Fabaceae, commonly known as
“grain legumes” or “pulses,” are major foodstuffs in most coun-
tries. In addition, these seeds may also provide some health benefits,
in particular in the area of hypercholesterolemia and hypertension
prevention. Whereas the hypocholesterolemic activity of soy
protein has been well known for decades and was finally supported
by the health claim by the U.S. Food and Drug Administration in
1999, similar information on non-soy legumes is scarce. This paper
reviews all such available data from animal models and human
trials as well as information on the mechanism of action provided
by in vitro studies, mainly on cell cultures or assays on specific
enzymes. This body of data indicates that a regular consumption
of grain legumes may be useful both for the prevention of hyperc-
holesterolemia and hypertension. More investigations are needed,
however, for elucidating the mechanism of action and the actual
effective components in legumes.
Keywords cardiovascular disease, cholesterol, coronary heart dis-
ease, hypertension, pea, plant protein, lupin
I. INTRODUCTION
The mature seeds of the plants of the Fabaceae, commonly
known as “grain legumes” or “pulses,” are major foodstuffs
in most countries and indispensable protein supplies for the
third world populations, especially for women and children. In
spite of the general consensus on the nutritional value of pulses
(Bouchenak and Lamri-Senhadji, 2013; Champ, 2002), their
consumption during the 20th century decreased in most indus-
trialized countries. This has been due to a number of reasons,
such as low digestibility, taste, and, of crucial importance, their
long cooking time, in a society where most people are able to
dedicate only a very short time to food preparation.
In the last decade, however, this scenario is slowly changing.
The growing awareness of the environmental impact of food
choices has generated a moderate increase in the consumption
of plant vs. animal foods (Reijnders and Soret, 2003). Whereas
only a minority of people continue to prefer a vegan or vegetar-
ian diet on the basis of philosophical or religious motivations,
more and more people are selecting a flexitarian diet (i.e., a
plant-based diet with the occasional inclusion of meat and fish
products), because they are looking for a healthier and more
sustainable lifestyle (Forestell et al., 2012).
Another stimulus for consuming more grain legumes derives
from the health benefits linked to their consumption, especially
in the area of dyslipidemia prevention. This was strongly val-
idated in 1999 when the U.S. Food and Drug Administration
(FDA) approved the “health claim” on the role of soy protein
in reducing the risk of coronary heart diseases (FDA, 1999),
mainly by lowering cholesterolemia. Most of the scientific lit-
erature in this area, is dedicated to soybean (Glycine max),
however, in the last 20 years, some papers have investigated
other legumes, in particular peanut (Arachis hypogea), chick-
pea (Cicer arietinum), lentil (Lens culinaris), white lupin (Lupi-
nus albus), narrow-leaf lupin (Lupinus angustifolius), yellow
lupin (Lupinus luteus), Andean lupin (Lupinus mutabilis), run-
ner bean (Phaseolus coccineus), Lima bean (Phaseolus lunatus),
common bean (Phaseolus vulgaris), pea (Pisum sativum), faba
bean (Vicia faba), cowpea (Vigna unguiculata), and mung bean
(Vigna radiata). This review will present and discuss the exper-
imental and clinical literature supporting the beneficial role of
the grain legumes in the prevention of hypercholesterolemia and
hypertension, which are the main risk factors of cardiovascular
disease that may be influenced by diet. Data on soybean will be
briefly summarized, since this legume has been the subject of
numerous reviews (Anderson et al., 1995; Harland and Haffner,
2008; Jenkins et al., 2010; Sacks et al., 2006; Sirtori et al.,
2007; Sirtori et al., 1998), whereas those of other legumes will
be discussed extensively, since this is the first comprehensive
review on this topic.
II. THE “BIG FOUR” FUNCTIONAL INGREDIENTS
FOR DYSLIPIDEMIA PREVENTION
Cardiovascular disease (CVD) is a major cause of death in the
Western populations and a constantly growing cause of mortal-
ity and morbidity worldwide (De Backer et al., 2003). It can be
prevented by lifestyle changes (such as diet, non-smoking, and
physical activity), which may reduce the risk of premature coro-
nary heart disease (CHD) by 82% (Stampfer et al., 2000); even
diet changes alone may reduce this risk by 60% (Kris-Etherton
et al., 2002). Epidemiological and clinical studies indicate that
a diet low in sodium and rich in fruits, vegetables, unrefined
grains, fish, and low-fat dairy products reduces the risk of CVD
and hypertension (Sirtori et al., 2009a).
Major risk factors for CVD include lipoprotein abnormal-
ities, elevated blood pressure, diabetes, smoking, and over-
weight. Commonly measures of lipoprotein status are serum
total cholesterol, low-density lipoprotein-cholesterol (LDL-C),
high-density lipoprotein-cholesterol (HDL-C), and triacylglyc-
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146 A. ARNOLDI ET AL.
TABLE 1
Hypocholesterolemic activity of soybean: changes in total cholesterol and LDL-C concentrations according to quartiles of the
study groups for initial cholesterol concentrations (Anderson et al., 1995)
Quartiles
Variable Q1 Q2 Q3 Q4
Total cholesterol
(mg/dL)
Initial range 127.1−197.8 201.2−255.4 259.3−332.8 >335
Change −5.2 −10.1 −22.2 −71.5
95% CI −17.1 to +6.7 −21.8 to +1.7 −37.3 to −7.1 −86.6 to −56.5
% Change −3.3 −4.4 −7.4 −19.6
LDL-C (mg/dL)
Change −7.1 −10.7 −18.3 −68.1
95% CI −20.0 to +6.0 −24.3 to +2.9 −35.3 to −1.3 −90.2 to −45.9
% Change −7.7 −6.8 −9.8 −24.0
erides (TAG). The changes of each lipoprotein are associated
with the following changes in the cardiovascular risk: +1%
total cholesterol =+2–3% risk; +1% LDL-C =+1.2–2.0%
risk; −1% HDL-C =+3% risk; whereas the risk change is not
well estimated for TAG (Anderson and Konz, 2001).
The CVD risk factors that are modifiable by diet, functional
foods, or dietary supplements are hypercholesterolemia, hyper-
triglyceridemia, diabetes, and hypertension. This section briefly
discusses the main functional components useful for improving
the lipid profile. They are generally indicated as the “big four”
and also include soybean protein.
A. Soybean Protein
Soy protein was shown to successfully reduce choles-
terolemia in experimental animals (Kim et al., 1980; Terpstra
et al., 1982), as well as in humans with cholesterol elevations
of genetic or non-genetic origin (Bakhit et al., 1994; Descovich
et al., 1980; Gaddi et al., 1987; Sirtori et al., 1977; Sirtori et al.,
1998). In addition, prospective observational studies, initially
in vegetarians (Burslem et al., 1978), then in Chinese women
(Zhang et al., 2003), and in a large population in Japan (Nagata
et al., 1998), have shown a reduction of total cholesterol and
LDL-C as well as of ischemic and cerebrovascular events with a
daily soy protein intake of more than 6 g, compared to less than
0.5 g/day. The 20-year follow-up of the Nurses Health Study
also indicated a significant correlation between vegetable pro-
tein intake and reduced cardiovascular risk (Halton et al., 2006).
The cholesterol lowering effect of soy proteins, potentially
leading to a reduced cardiovascular risk, became the basis for
the U.S. Food and Drug Administration (FDA) approval of the
health claim for the role of soy protein consumption in coronary
disease risk reduction (FDA, 1999).
In the earliest studies in the 1970s, a soy protein prepara-
tion, given to hospitalized highly hypercholesterolemic patients,
was found to be highly effective for cholesterol lowering and
well tolerated. In this six-week controlled crossover investiga-
tion (Sirtori et al., 1977), there was a 20–22% reduction in total
cholesterol level and a 22–25% reduction in LDL-C, without sig-
nificant changes of triglyceridemia. The cholesterol reduction
was inversely related to baseline cholesterolemia and not mod-
ified by the addition of dietary cholesterol (Sirtori et al., 1977).
The numerous ensuing clinical studies were summarized in
a milestone meta-analysis of 38 studies, in both hypercholes-
terolemic and normolipidemic individuals (Anderson et al.,
1995). Dividing the studies in four quartiles depending on base-
line cholesterolemia (Table 1), this meta-analysis showed that
the serum LDL-C concentrations are modified from a minimum
of −6.8% in subjects with mild cholesterolemia (initial total
cholesterol value in the range 200–255 mg/dL), up to −24%
in case of severe hypercholesterolemia (initial total cholesterol
value >335 mg/dL). Most of the variance of the net changes
in cholesterol concentrations depends on the square of initial
cholesterol value, whereas other variables, such as the kind of
soy products and its amounts, the type of diet and the age, give
only marginal contribution to the variance.
A subsequent review (Sacks et al., 2006) has criticized the re-
sults of this meta-analysis, since the studies published in the fol-
lowing years appeared not to confirm the very powerful choles-
terol reducing effect of soy proteins. Another more recent review
has shown, however, that the net cholesterol reductions observed
in recent investigations are comparable with those of the studies
included in the Anderson meta-analysis, when they are located
in the correct quartiles of Table 1 (Sirtori et al., 2007). The
apparent large differences between recent and old studies de-
pend, instead, on the fact that about 25% of older studies were
based on severely hypercholesterolemic individuals, whereas in
recent years patients with cholesterolemia in the very high range
(>335 mg/dL) are no more eligible for dietary intervention stud-
ies, since they are now treated with specific drugs.
A subsequent systematic review and meta-analysis has
considered all trials reporting association between a moderate
daily intake of soy protein and blood cholesterol (Harland and
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GRAIN LEGUMES 147
Haffner, 2008). Thirty studies containing 42 treatment arms
(n=2913), with an average soy protein intake of 26.9 g,
met the inclusion criteria. The soy protein treatment led to
reductions in mean total cholesterol, LDL-C, and blood TAG
of 0.22 mmol/L (95% CI −0.142 to −0.291, p<0.0001),
0.23 mmol/L (95% confidence interval, CI −0.160 to −0.306,
p<0.0001), and 0.08 mmol/L (95% CI −0.004 to −0.158,
p=0.04), respectively. This meta-analysis concluded that a
modest amount of soy protein (ca. 25 g) into the diet of adults
with normal or mild hypercholesterolemia results in small, but
highly significant, reductions in total and LDL-C, equivalent to
ca. 6% LDL-C reduction.
B. Soluble Fiber
Dietary fiber is recommended as a safe and practical ap-
proach for cholesterol reduction. Whereas insoluble fibers (cel-
lulose +lignin) are almost inactive (Anderson, 2000), a reg-
ular consumption of soluble viscous fibers may result in a
cholesterol-lowering effect in the order of 5–10% (Jenkins et al.,
2000). The soluble fibers that effectively lower LDL-C, include
apple, lemon and orange pectins (Brouns et al., 2012; Gonz´
alez
et al., 1998), oat (Ruxton and Derbyshire, 2008) and barley β-
glucan (Keenan et al., 2007), psyllium (Shrestha et al., 2006)
and flaxseed (Erkkil¨
a and Lichtenstein, 2006) fibers. Addition-
ally, a paper suggests a potential application of narrow-leaf lupin
fiber (Hall et al., 2005a). The mechanisms of these effects are
likely to be multifactorial. An important potential mechanism
is the binding of cholesterol and bile acids by the fiber that im-
pairs their absorbance in the gut (Flight and Clifton, 2006). In
addition, inhibition of cholesterol synthesis by short-chain fatty
acids, produced by fermentation of the fiber in the colon, has also
been hypothesized (Hara et al., 1999). Based on clinical study
evidence, oat soluble fiber was approved as hypocholesterolemic
ingredient in the European Union and the United States.
Fasting and post-prandial TAG responses may depend upon
the fiber content of the diet the carbohydrate availability. High-
carbohydrate/low-fiber diets increase fasting serum TAG (Park
and Hellerstein, 2000), whereas high-carbohydrate/high-fiber
diets reduce TAG (Anderson, 2000; Anderson and Ward, 1979).
The comparison of a high-fiber Mediterranean diet with a
low-carbohydrate diet for weight management (Shai et al.,
2008) led to similar weight losses, but the high-fiber diet pro-
vided more favorable effects on lipids (LDL-C change −5.6%
with the high-fiber diet vs. −3.0% with the low-carbohydrate
diet).
The fiber content can influence the food glycemic index (GI).
The GI values of legumes are lower than that of cereals. This
is relevant, since diets with a lower glycemic index are asso-
ciated with significantly higher HDL-C values vs. diets with a
higher GI (Anderson et al., 2004; Frost et al., 1999). A low GI
diet, i.e., based on low GI breads, cereals, pasta, parboiled rice,
beans, peas, and nuts, increased HDL-cholesterol by 1.7 mg/dL
compared to a reduction of 0.2 mg/dL in the high-carbohydrate /
low-fiber diet with a similar caloric intake (Jenkins et al., 2008).
C. Phytosterols
All plants produce sterol compounds (phytosterols), struc-
turally related to cholesterol. Experimental and clinical studies
have demonstrated that high phytosterol intakes (1–2 g) reduce
the intestinal absorption of dietary and biliary cholesterol. A
meta-analysis (Demonty et al., 2009) on 84 trials including
141 trial arms has established a pooled reduction of LDL-C
of 0.34 mmol/L, i.e., 8.8%, for a mean daily dose of 2.15 g
phytosterols. As in the case of protein and soluble fiber, the
effect is enhanced in subjects with high baseline LDL-C levels.
Phytosterols interfere with the micellar solubilisation of choles-
terol in the intestine thus reducing the efficiency of cholesterol
absorption (Genser et al., 2012). The final outcomes are alter-
ations in serum lipoproteins with significant decreases in LDL-
C concentrations (Davidson et al., 2001; Gylling et al., 1997;
Hallikainen and Uusitupa, 1999; Maki et al., 2001; Matvienko
et al., 2002; Miettinen et al., 1995; Neil et al., 2001; Ntanios
and Duchateau, 2002). The U.S. Food and Drug Administration
and the European Commission have approved the health claim
on phytosterols (1–2 g per day) and cholesterol reduction.
The average phytosterol intake from plant foods in adults is
generally in the range of 160–350 mg/day. It seems feasible that
a prolonged consumption of this small amount of phytosterols
may contribute to the prevention of hypercholesterolemia, even
if it does not significantly alter the cholesterol level in short-time
experiments.
D. Omega-3 Fatty Acids
Omega-3 Fatty acids are very relevant for CVD prevention.
The main mechanism underlying their preventive activity is the
reduction of serum TAG, however other functions are impor-
tant, i.e. antithrombotic activity (reduced platelet aggregation),
anti-inflammatory activity, modulation of the endothelial func-
tion, antiarrhythmic activity, and their capacity for stabilizing
the atheromatous plaque (Sirtori et al., 2009b). However, most
of these activities are exerted by omega-3 long chain polyun-
saturated fatty acids (LCPUFA), e.g., eicosapentaenoic acid
(EPA, 20:5 omega-3) and docosahexaenoic acid (DHA, 22:6
omega-3), provided in the diet almost exclusively by animal
foods (especially fish) or seaweeds. Plant foods (for example
spinach), certain seeds (such as flaxseed, walnut, rapeseed, soy-
bean, and white lupin) and their extracted oils contain, however,
α-linolenic acid (ALA, 18:3 omega-3), the metabolic precursor
of long chain omega-3 fatty acid. In all cases (with the exception
of flaxseed), the ALA contents are lower than those of linoleic
acid, the corresponding omega-6 fatty acid.
Although the role of ALA in cardiovascular prevention
has been rarely investigated, some epidemiological (Djouss´
e
et al., 2005) and a few mechanistic studies (Rallidis et al.,
2004; Zhao et al., 2004) suggest a potential CVD protection
by ALA. A UK Food Safety Authority Workshop, however,
considered this evidence still equivocal (Sanderson et al.,
2002) and a review (Wendland et al., 2006) concluded that
most cardiovascular risk markers do not appear to be affected
by ALA consumption, even if ALA supplementation causes
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148 A. ARNOLDI ET AL.
TABLE 2
Systematic names, common names and proximate analyses (% on dry weight) of main grain legumes (adapted from Belitz et al.,
2009)
Systematic name Common name Crude protein Lipids
Digestible
carbohy-
drates Crude fibre Ash
Arachis hypogea Peanut, groundnut 27.450.79.17.52.7
Cicer arietinum Chickpea 22.75.054.610.73.0
Glycine max Soybean 39.019.67.616.65.5
Lens culinaris Lentil 28.61.657.611.93.6
Lupinus albus White lupin 44.712.513.638.35.0
Lupinus angustifolius Narrow-leaf lupin 31.16.05.314.73.5
Lupinus luteus Yellow lupin 50.26.18.430.74.1
Lupinus mutabilis Andean lupin, pearl lupin,
tarwi
42.724.011.06.84.7
Phaseolus coccineus Runner bean, scarlet runner
bean
23.12.155.24.53.9
Phaseolus lunatus Lima bean, butter bean 25.01.657.815.03.9
Phaseolus vulgaris Common bean, garden bean
(pinto)
24.11.854.119.24.4
Pisum sativum Pea 25.71.453.718.73.0
Vicia faba Broad bean, faba bean 25.71.546.510.35.6
Vigna radiata Mung bean, green gram 26.91.646.35.13.6
Vigna unguiculata Cowpea 24.12.363.314.12.9
small decreases in fibrinogen concentrations and fasting plasma
glucose, which are other risk factors for CVD. The fact that
the available evidence is still insufficient to recommend the
supplementation with ALA to reduce CVD does not limit the
importance of ALA in a healthy diet.
III. NUTRITIONAL COMPONENTS OF GRAIN
LEGUMES RELEVANT IN DYSLIPIDEMIA
PREVENTION
The systematic and common names and the average proxi-
mate analyses of major grain legumes are shown in Table 2. On
the basis of their composition, the grain legumes listed in this
table may be roughly divided into three classes: in soybean and
lupin the most abundant components are the proteins (>35%);
in peanut the lipids >50%); and in all other legumes digestible
carbohydrates (>45%). This section will describe in brief the
legume components that are relevant for hypercholesterolemia
prevention, i.e., proteins, soluble fibers, and lipids. A recent
review has extensively discussed the role of grain legume phy-
tochemicals, such as isoflavones, polyphenols, and saponins,
in the prevention of the cardiometabolic risk (Bouchenak and
Lamri-Senhadji, 2013). Another review, instead, has considered
only isoflavones (Sirtori et al., 2005).
A. Proteins
Legume proteins are considered important in vegetarian diets
or diets poor in animal proteins, because they have a satisfactory
content of lysine and other indispensable amino acids, but have
a slightly insufficient content of sulphur amino acids as their
main defect. The fractionation by differential solubility sepa-
rates legume proteins in albumins, globulins, prolamins, and
glutelins: globulins are predominant, whereas prolamins and
glutelins are very scarce or absent. Legume globulins, which
function mostly as storage-proteins, to be mobilized during the
course of germination, can be further separated by ultracentrifu-
gation or chromatography into the following two major compo-
nents: legumins (11S) and vicilins (7S), and some additional
other minor fractions. The relative abundance of these legume
fractions depends on the species.
In soybean, legumins (also referred to as glycinin) are de-
rived from a protein precursor, which is split into an acidic
polypeptide A (pI ≈5) and a basic polypeptide B (pI ≈8.2).
These two peptides are linked by a disulfide bridge and are
considered as subunits. Strong homology exists between the
legumins of different legume species and the A/B polypeptide
cleavage site is highly conserved, although variable regions are
found mostly in the A peptide. Generally, legumins are not gly-
cosylated, but there are exceptions, such as lupin α-conglutin.
Vicilins derive from the post-translational cleavage of a single
precursor into some polypeptides. Soy vicilin (also referred to
as β-conglycinin) consists in the aggregation of three polypep-
tides, named α,α, and β, not linked by disulfide bridges.
These trimers may contain either identical or different polypep-
tides (hetero- and homopolymeric forms). In all legumes, the
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GRAIN LEGUMES 149
sequences of precursor proteins are highly homologous, but the
cleavage sites are variable. The vicilins are in general glycosy-
lated, but at a different extent in each legume.
In general, under non-denaturating conditions, the legumins
and vicilins exhibit a tendency towards reversible dissocia-
tion/association, depending mostly on the pH value and ionic
strength. Legumins are relatively more stable than vicilins. The
lower stability of the latter proteins is evident also during in-
dustrial processing. For example, the comparison of the na-
tive globulins of soybean (Gianazza et al., 2003), narrow-leaf
lupin (Sirtori et al., 2010a) and pea (Sirtori et al., 2012b) with
some industrially processed protein isolates performed by 2-D
electrophoresis and mass spectrometry has shown that most of
the spots of the processed materials derive from the legumins,
whereas the spots of the vicilins are scarce. The instability of
vicilins to processing may be important in understanding the
CVD protective effects of commercial legume protein isolates,
since the αsubunit of vicilin is considered the major hypoc-
holesterolemic component of soybean protein (Duranti et al.,
2004) and its breakdown may affect its bio-activity.
B. Carbohydrates
Most grain legumes contain 39–51% starch, with the excep-
tion of peanut, soybean, and lupin. Legume starch has a higher
content of amylose (30–45%) than cereals and potato (0-30%).
The consequences are the following : i) a higher gelatinisation
temperature of starch granules, and ii) a higher susceptibility to
retrodegradation with the consequence of reducing digestibility
by endogenous amylases (Guillon and Champ, 2002). Partially
undigested starch (resistant starch) reaching the large intestine is
fermented by the colonic microbiota (Finley et al., 2007). The
fermentation of resistant starch produces short chain organic
acids, such as butanoate, a main colonocyte nutrient and poten-
tially involved in the protection of colon cells (Bird et al., 2000).
Grain legumes are also a source of non-starch polysaccha-
rides (NSP). For example, lentils and common beans contain
10.6 and 17.3% NSP (on dry seed), 12.4 and 26.3%, respec-
tively, being soluble NSP. They are located both in the seed
hull, which contains mostly insoluble NSPs, such as cellulose,
and in the endosperm cell walls, which are richer in soluble
fibers, such as pectic polysaccharides. These soluble fibers may
decrease the digestibility of starch contributing the low GI of
starchy legumes (Guillon and Champ, 2002); varying between
18 and 56, compared to GI values of cereal based foods, which
generally fall in the range 65–95, but may be even greater than
100 in some cases (http://www.glycemicindex.com). The ad-
dition of lupin flour in white wheat bread has been shown to
decrease its GI (Hall et al., 2005b). Low GI diets are benefi-
cial in diabetes (Fatima and Kapoor, 2006) and in general may
assist in the prevention of cardiovascular disease. For example,
a decrease of the GI of the diets of hyperlipidemic patients re-
sulted in a significant reduction in total cholesterol, LDL-C and
TAG, compared with values during the preceding and following
months (Jenkins et al., 2000).
Low molecular weight indigestible carbohydrates, such as
α-galactosides, are usually considered as responsible for the
flatus, however, they may provide beneficial effects, since in
the human colon they can stimulate the growth and activity of
beneficial bifidobacteria and lactobacilli (Mart´
ınez-Villaluenga
et al., 2008). Specific methods have been developed for the
purification of these α-galactosides from different legumes, for
example from the lupin seed (Mart´
ınez-Villaluenga et al., 2004).
C. Lipids
Most legume seeds contain only small quantities of lipids,
but a few contain much more, i.e., peanuts, soybeans, and white
lupin (Table 2). The composition of peanut is more similar to tree
nuts than to legumes and this seed is mainly used for producing
peanut oil and butter. The production of oil from soybean also
has a considerable economic impact in many countries, whereas,
to our knowledge, lupin oil is not yet produced commercially.
Table 3 shows the fatty acid composition of these legumes. From
a nutritional point of view, the composition of white lupin oil
appears very promising, because it is in line with recent dietary
recommendations (Simopoulos, 2006) for CVD prevention (>
50% oleic acid, <18% linoleic acid, and >7% α-linolenic acid).
The phytosterol content of raw (Ryan et al., 2007) and
cooked legumes (Kalogeropoulos et al., 2010) has been recently
TABLE 3
Percent fatty acid composition of the oils of selected grain legumes (adapted from Belitz et al., 2009)
Fatty acid L. albus (%)
L. angustifolius
(%) L. luteus (%) L. mutabilis (%) G. max (%) A. hypogea (%)
16:0 7.811.04.89.810 10
18:0 1.63.82.57.85 3
18:1 53.038.221.053.921 41
18:2 (ω-6) 17.237.147.525.953 35.5
18:3 (ω-3) 9.55.37.52.6 8 trace
20:0 & 20:1 5.51.24.50.64 2.5
22:0 & 22:1 5.81.97.90.50 3
ω-6/ω-3 1.81 7.01 6.33 20.36.63 >100
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150 A. ARNOLDI ET AL.
TABLE 4
Percent coverage of recommended daily intakes for selected macro and micronutrients by consuming one serving of cooked
legumes (125 g) (adapted from Kalogeropoulos et al., 2010)
Percent coverage of recommended daily intakes
Protein Dietary fiber Phytosterols ALAb
Polyphenols
(aglycones)
Cicer arietinum 10.640.724.45.548.7
Lens culinaris 10.738.311.42.226.8
Lupinus albus 15.814.526.835.611.5
Phaseolus vulgaris 10.637.516.421.54.0
Pisum sativum 9.339.116.915.75.4
Vicia faba 8.823.017.54.016.6
Vigna unguiculata 9.627.06.723.916.5
Recommended
Daily Intakesa
91 g 31 g 250 mg 1700 mg 23-28 mg
(a) USDA Food Guide (http://www.cnpp.usda.gov/DietaryGuidelines.htm).
(b) ALA: alpha-linolenic acid.
investigated. In most legumes the main phytosterol is β-
sitosterol, accompanied by small quantities of campesterol, 5-
avenasterol, and stigmasterol, with quantities ranging from 13-
53 mg/100 g fresh weight in cooked dry legumes (Table 4).
The lipid fraction contains other lipophilic nutrients, such as
tocopherols. The most abundant tocopherol in legumes is usu-
ally γ-tocopherol, followed by minor quantities of α-tocopherol
(with the exception of common bean lacking this isomer), and
δ-tocopherol (with the exception of narrow-leaf lupin and An-
dean lupin). However, β-tocopherol, the most active form of
vitamin E, and tocotrienols are both absent from grain legumes.
Containing more than 10 mg/100 g seeds of total tocopherols,
soybean, pea, white lupin, and chickpea may significantly con-
tribute to the daily intake of this vitamin (Boschin and Arnoldi,
2011); however, the long cooking required before their con-
sumption may partly destroy tocopherols (Kalogeropoulos et al.,
2010).
D. Final Considerations
Whereas most of the available literature reports the compo-
sition of raw seeds, a recent paper has investigated the resid-
ual amounts of bioactive constituents remaining after standard
cooking of some common legumes (Kalogeropoulos et al.,
2010). Table 4 presents a selection of the results of this paper.
One serving of each legume, corresponding to 125 g, provides
up to 15 g protein (15% recommended daily intake), 12 g di-
etary fiber (40% recommended daily intake), 67 mg phytosterols
(26% recommended daily intake), 590 mg ALA (35% recom-
mended daily intake), and 12 mg polyphenols (48% recom-
mended daily intake). All of these components may contribute
to the capacity of legumes to prevent cardiovascular disease
each through different mechanisms, possibly acting in synergy.
IV. HYPOCHOLESTEROLEMIC ACTIVITY OF GRAIN
LEGUMES
A. Animal Studies
The first studies on the potential hypocholesterolemic effects
of soybean were performed in the ‘70s, whereas it was not until
the ‘90s that some researchers started to dedicate resources and
time to non-soy legumes, stimulated by the similarities among
the sequences of the main protein fractions in legumes. Most of
the experimentation has been performed using the rat model of
hypercholesterolemia, i.e., feeding rats a diet rich in a saturated
oil and containing 1–2% cholesterol and 0.5% cholic acid (Nath
et al., 1959) and using casein or lactalbumin as control proteins.
This model, initially developed for drug evaluation, is the least
expensive one for these kinds of studies. Some researchers,
instead, prefer to use more expensive animal models, such as
hamster, pig, or rabbit, for specific purposes.
Table 5 reports the experimental design (animal model, du-
ration, kind and amount of legume ingredient) and the results
of 16 animal model investigations performed on grain legumes
and two relevant studies on soy protein that will be discussed in
detail in section IV-C of this review. Initially, most studies were
performed on the whole kernel flour (raw or cooked to decrease
the content of anti-nutritional factors), whereas in recent years
the investigations on protein concentrates or isolates prevail,
especially in the case of lupin and pea.
1. Chickpea, cowpea, common bean, faba bean, lentil and
pea
A report compared the hypocholesterolemic effects of diets
containing four legumes: lentil, pea, common bean, and Lima
bean (Dabai et al., 1996). All experimental diets were effective
at lowering cholesterol levels, but the diets containing common
bean and Lima bean were more potent than the diets based on
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TABLE 5
Experimental studies on rat, hamster or rabbit fed grain legumes: design, main features, and effects on lipid metabolism (nr =not reported)
Total cholesterol
(VLD-C +LDL-C) or
non-HDL-C HDL-C TAG
Reference Grain legume Tested ingredients Animal
Duration
(days)
Ingredient in
diet (%)
Chol. in the
diet (%)
Control
value
(mmol/L)
Change vs.
control (%)
Control
value
(mmol/L)
Change vs.
control (%)
Control
value
(mmol/L)
Change vs.
control (%)
Control
value
(mmol/L)
Change vs.
control (%)
Fukui et al., 2002 G. max Crude protein Rat 14 20 0.53.8−27.1nr nr 1.3−32.01.7−18.2
Fukui et al., 2002 G. max Purified protein Rat 14 20 0.53.8−22.7nr nr 1.3−30.01.7−10.3
Duranti et al.,
2004
G. max 7S globulin Rat 28 200 mg/kg
body weight
17.8−49.0nr nr nr nr 2.7−50.0
Duranti et al.,
2004
G. max α7S glubulin Rat 28 20 mg/kg
body weight
17.8−36.0nr nr nr nr 2.7−34.0
Zulet et al., 1999 C. arietinum Cooked flour Rat 16 70 1 3.6−34.12.27 −43.20.90 −17.82.4−20.3
Dabai et al., 1996 L. culinaris Cooked flour Rat 56 33 1 6.7−6.74.87 −32.20.98 −2.00.8−33.3
Chango et al.,
1998
L. albus Whole flour Rat 28 45 1 2.5−17.8nr nr nr nr 1.1−49.1
Sirtori et al., 2004 L. albus Protein isolate Rat 21 50 mg/kg
body weight
15.6−22.74.90 −30.20.70 +29.60.84 −16.2
Bettzieche et al.,
2008
L. albus Protein isolate Rat 20 5 1 4.1−11.21.39 −38.71.1+40.31.1−43.8
Guadagnucci
Fontanari
et al., 2012
L. albus Cooked flour Hamster 28 55 1 6.2−16.83.48 −43.42.7+16.91.4+22.1
Guadagnucci
Fontanari
et al., 2012
L. albus Purified protein Hamster 28 22 1 6.2−15.33.5−28.62.73 +1.71.4−25.1
Marchesi et al.,
2008
L. albus Protein isolate Rabbit 90 20 1 48.6−33.547.1−34.61.55 +0.113.6−15.8
Bettzieche et al.,
2008
L. angustifolius Protein isolate Rat 17 5 1 8.9+9.72.4−13.11.93 +1.01.1−2.8
Bettzieche et al.,
2008
L. angustifolius Vicilin +legumin Rat 17 5 1 8.9−5.12.4−19.21.93 +33.71.1−20.6
Parolini et al.,
2012
L. angustifolius Protein isolate Rat 28 20 1 8.0−55.37.8−61.10.58 +20.00.55 −10.4
Chango et al.,
1998
L. luteus Whole flour Rat 28 40 1 2.5−11.3nr nr nr nr 1.1−12.3
Lakesan et al.,
1995
P. sativum protein Rat 28 24 1 4.1−26.9nr nr 1.17 −57.78 1.8−40.1
Dabai et al., 1996 P. sativum Whole cooked
flour
Rat 56 33 1 6.7−14.04.9−26.90.98 −8.16 0.82 −11.4
Spielmann et al.,
2008
P. sativum Ethanol washed
protein
Rat 16 20 0 1.7−2.90.9−5.91.06 +5.71.9−1.1
Rigamonti et al.,
2010
P. sativum Protein isolate Rat 28 20 1 7.5−51.67.1−58.10.43 +54.80.93 −39.0
Dabai et al., 1996 P. lunatus Whole cooked
flour
Rat 56 33 1 6.7−24.04.9−38.80.98 +40.82 0.82 −2.8
Dabai et al., 1996 P. vulgaris Whole cooked
flour
Rat 56 33 1 6.7−36.34.9−53.00.98 +41.80.82 −39.1
Macarulla et al.,
2001
V. faba Whole flour Rat 14 68 1 3.5−36.82.5−56.30.92 +3.26 1.2−39.2
Macarulla et al.,
2001
V. faba Protein isolate Rat 14 23 1 3.5−29.92.5−36.60.92 −9.78 1.2−10.8
Frota et al., 2008 V. unguiculata Cooked flour Rat 28 20 1 4.6−48.52.1−54.22.58 −44.10.88 −4.5
Frota et al., 2008 V. unguiculata Purified protein Rat 28 20 1 4.6−19.42.1−18.42.58 −20.10.88 +24.1
151
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152 A. ARNOLDI ET AL.
lentil and pea (Table 5). The better cholesterol-lowering capacity
of the legume diet was not associated with larger concentrations
of faecal bile acids or neutral sterols.
Another report compared lentil, pea, common bean, and Lima
bean using the pig model (Kingman et al., 1993). Thirty-six
growing boars were randomly allocated in groups to six differ-
ent diets. The diets were: 1) a semi-purified (SP; control group
1) diet; 2) SP +10 g cholesterol/kg (SPC; control group 2); 3),
4), 5), and 6) SPC +cooked legumes [70:30, w/w; lentil, pea,
Lima bean, and common bean, respectively]. After 42 d, the
diet-induced hypercholesterolemia was significantly inhibited
in the groups consuming pea, common bean, and Lima bean,
although HDL-C levels were maintained (data not reported in
Table 5). This paper also provided interesting mechanistic data:
the faecal steroid excretion by the legume groups was not signif-
icantly different from that of control group 2, suggesting that the
mechanism for the hypocholesterolemic activity did not involve
increased hepatic bile acid synthesis and thereby an increased
cholesterol clearance via the intestinal route.
In the same animal model, another report showed that the
whole seed flour of chickpea was able to significantly improve
the lipoprotein profile vs. the control diet (Zulet et al., 1999).
Another report compared the hypocholesterolemic activity
of the whole seed flour and a protein isolate (prepared by iso-
electric point precipitation and spray drying) of faba bean vs.
casein (Macarulla et al., 2001). Both faba bean diets signifi-
cantly lowered body weights and energy intakes vs. casein, but
the whole seed was more effective than the protein isolate diet
in improving the lipoprotein profile. Liver cholesterol and TAG
were also reduced in both diets.
A comparison of cowpea whole seed flour vs. protein iso-
late effects on cholesterolemia using a hamster model (Frota
et al., 2008) produced equivalent results to those of faba bean
(Macarulla et al., 2001). Both cowpea diets were effective, but
the improvement of the lipoprotein profile induced by the whole
seed flour was more favourable. In addition, a histological study
of the liver showed that both diets reduced liver steatosis (i.e.,
accumulation of fat in the liver) by more than 75%.
The hypercholesterolemic rat model has also been used to
study pea. In the earliest reported study, the rat diet contained
20% pea protein or casein (Lasekan et al., 1995): the pea protein
reduced cholesterol and TAG by 27% and 40% respectively.
Plasma glucose, insulin, and the apo Al levels were slightly
lower in rats fed pea proteins vs. those fed casein. Similar results
were also obtained in the study already cited in section IV.A.1,
in which pea was compared with other legumes (Dabai et al.,
1996).
An investigation (Spielmann et al., 2008) aimed to compare
the potential hypocholesterolemic effect of a pea protein con-
centrate vs. casein gave only partially positive results. In this
case, the hypercholesterolemic diet raised the baseline values
of total cholesterol and LDL-C much less than it would be ex-
pected in this kind of studies. Therefore, some differences in the
protocol may be responsible of the out-come. However, the effi-
cacy of the pea protein treatment was confirmed by the decrease
of very low density lipoprotein (VLDL) cholesterol.
A recent paper has shown that a diet containing a commer-
cial pea protein isolate (PisaneTM) significant decreased total
cholesterol and TAG when compared to a casein diet (Riga-
monti et al., 2010) and this study also provided some insight in
the mechanism of action. Pea protein-fed rats displayed higher
hepatic mRNA levels of the LDL receptor vs. those fed casein
(p<0.05), whereas the mRNA concentration of genes involved
in fatty acids synthesis, such as fatty acid synthase and stearoyl-
CoA desaturase, was lower in pea protein-fed rats (p<0.05).
These results indicate that the pea protein affects cellular lipid
homeostasis by up-regulating genes involved in hepatic choles-
terol uptake and by down-regulating fatty acid synthesis genes.
2. Lupin
The generic term lupin includes four domestic species: L. al-
bus (white lupin), L. angustifolius (narrow-leaf lupin), L. luteus
(yellow lupin), and L. mutabilis (Andean lupin). Whereas the
last has not yet attracted the research interest in this field and
yellow lupin has been investigated only once, white lupin and
narrow-leaf lupin have been extensively investigated by differ-
ent research groups and using different models.
The cholesterol modulating properties of the whole seed
flours of yellow and white lupin were first investigated 20 years
ago in rats (Chango et al., 1993), observing small but significant
decreases of total serum cholesterol and TAG levels: however,
from this study it was not possible to conclude what were the
responsible seed components.
Around 10 years later, a total protein extract (TPE) from
white lupin was investigated using a pharmacological approach
(Sirtori et al., 2004). Rats were fed a cholesterol-rich diet con-
taining 20% casein and treated daily by gavage with 50 mg/rat
of TPE for 14 days vs. the vehicle only. The lupin-treated rats
showed significant decreases in total cholesterol (22.7%), LDL-
C (30.2%), and TAG (16.2%), whereas glucose was unaffected.
It is important to note that in this experiment the dose given to
animals was only 50 mg/kg body weight per day.
A few years later, the availability of large amounts of a
purified protein isolate from white lupin (D’Agostina et al.,
2006) containing mostly legumins +vicilins (W-LUP), pre-
pared by the Fraunhofer Institute IVV (Freising, Germany),
allowed further advancement in the understanding of the CVD
protective effects of lupin. The cholesterol-lowering activity
of this material was reported in the rat model of hyperc-
holesterolemia (Bettzieche et al., 2008b), whereas the poten-
tial anti-atherosclerotic activity was tested in a rabbit model of
atherosclerosis (Marchesi et al., 2008). In this rabbit animal
a focal plaque development at both common carotid arteries is
induced by perivascular injury (Chiesa et al., 2001). After recov-
ery from surgery, for 90 d animals were fed three different diets,
all containing 1% cholesterol, 15% saturated fatty acids and
20% protein: the protein sources were casein (control), W-LUP,
or a 1:1 mixture of casein +W-LUP. Lower cholesterolemia
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GRAIN LEGUMES 153
was detected in the W-LUP vs. the casein group at 60 and 90
d of treatment (−40.3% and −33.5%, respectively; p<0.05).
Cryosection analyses of the carotids indicated a significant re-
duction in focal lesion progression in the W-LUP vs. the casein
group (−37.4%; p<0.05). This important study showed that
white lupin protein not only reduces cholesterolemia, but also
exerts a protective activity against atherosclerosis progression.
The same model had been previously applied to demonstrate
hypocholesterolemic and anti-atherosclerotic activity of soy-
bean protein (Castiglioni et al., 2003).
A total protein extract from white lupin was recently tested
in a hamster model of hypercholesterolemia (Fontanari et al.,
2012). Interestingly, the lupin diet fed hamsters had a low level
of liver steatosis (accumulation of fat in the liver) as compared
to those fed the casein-diet (control diet).
More recently, also narrow-leaf lupin has attracted the re-
search interest. An investigation (Bettzieche et al., 2008a) com-
pared the hypocholesterolemic activity of the total seed protein
extract with a purified fraction containing mostly legumins +
vicilins (NL-LUP) (again prepared by the Fraunhofer Institute
IVV). The NL-LUP was more effective than the total protein
extract in lowering cholesterol.
In a second study (Parolini et al., 2012), the treatment with
NL-LUP markedly lowered plasma total cholesterol level com-
pared to casein (-55.3%, p<0.0005), whereas no significant
differences were observed for TAG and HDL-C levels between
the two groups. The lupin-fed rats also displayed higher hepatic
mRNA levels of SREBP-2, a major transcriptional regulator of
intracellular cholesterol levels, and CYP7A1, the rate-limiting
enzyme in bile acid biosynthesis (p<0.05).
B. Human Studies
Only a few publications have reviewed the clinical studies on
the cholesterol lowering effects of legumes, in combination with
other health benefits (Bouchenak and Lamri-Senhadji, 2013).
We recommend in particular two meta-analyses (Anderson and
Major, 2002; Bazzano et al., 2011), both indicating that a diet
rich in grain legumes is able to decrease total cholesterol and
LDL-C. Both meta-analyses discussed studies performed on
the whole seeds of a single grain legume or two or more grain
legumes in combination.
A thorough literature research using PubMed and Web of Sci-
ence has provided 19 papers dealing with legumes and choles-
terol, giving a total of 21 different trial arms, which are listed in
Tables 6 and 7. These studies may be split into four groups: 1)
twelve unblind studies on whole seeds; 2) one unblind study on
a model beverage from white lupin seed; 3) two blind studies
on model foods containing whole kernel flours; 4) four blind
studies on purified fiber or proteins. Table 6 reports the main
features of these dietary interventions, whereas Table 7 gives
their design and main outcomes.
Considering gender, nine studies are on both genders, nine
on males alone, and, surprisingly, none on females alone. The
tables include two uncontrolled studies (Jenkins et al., 1983;
Nowicka et al., 2006), which compared the effects of mixed
beans, in the former case, and a drink based on white lupin, in
the latter, with the preceding lipid levels of stabilized patients.
It was decided to include these uncontrolled studies, since each
one can be considered a pioneer investigation: the former was
the first on grain legumes and cholesterol, the latter was the first
on a model food obtained from a legume.
All 17 controlled studies are randomized: seven have a par-
allel design, whereas ten have a crossover design. The durations
span from three weeks to one year, but a four-week duration is
prevalent. Only a few studies report the total cholesterol/HDL-C
and/or the LDL-C/HDL-C ratios (see Table 7).
Four studies of group 1 are on common bean (Anderson
et al., 1990; Anderson et al., 1984; Oosthuizen et al., 2000;
Winham and Hutchins, 2007), in one case in combination with
oat (Mackay and Ball, 1992), one is on chickpea (Pittaway
et al., 2008), one is on peanut (Nouran et al., 2010), the others
are on mixed legumes (Abeysekara et al., 2012; Duane, 1997;
Hermsdorff et al., 2011; Jenkins et al., 1983). The quantity
of legumes consumed daily varies as well as the number of
servings per week, which spans from 4 to 7 servings. Eight
out of these twelve studies showed significant decreases in total
cholesterol and/or LDL-C in the range from −8to−56 mg/dL
(Abeysekara et al., 2012; Anderson et al., 1990; Anderson et al.,
1984; Duane, 1997; Hermsdorff et al., 2011; Jenkins et al., 1983;
Pittaway et al., 2008; Winham and Hutchins, 2007) and in a few
cases also in TAG (Anderson et al., 1990; Jenkins et al., 1983).
The significant changes in total cholesterol and LDL-C are in
the range from -7.5 mg/dL to -56 mg/dL. In a few cases, also the
changes of total cholesterol/HDL-C and/or the LDL-C/HDL-C
ratios are significant (Mackay and Ball, 1992; Nouran et al.,
2010; Winham and Hutchins, 2007).
Group 2 contains only an uncontrolled three-month study
(Nowicka et al., 2006) that evaluated the effect of a daily in-
take of 500 mL of a model drink obtained from white lupin
seed on male and female smokers. Significant decreases in to-
tal and LDL-C were observed in respect to the stabilised lipid
values during the previous diet. Interestingly this investigation
produced also decreases of blood pressure (see section V.A.2).
Group 3 includes two blind studies on model foods contain-
ing whole kernel flour. The former (Fr¨
uhbeck et al., 1997), on
faba bean flour mixed to smashed potato, reports segregated re-
sults for normocholesterolemic patients (mean initial cholesterol
equal to 200 mg/dL) and moderate hypercholesterolemic (mean
initial cholesterol equal to 240 mg/dL). Both groups of patients
showed decreases of total and LDL-C that were higher in the
hypercholesterolemic group. However, no significant changes
were observed in the latter study (Belski et al., 2011), based
on normolipidemic subjects who consumed bread, biscuits and
pasta added with lupin flour.
The blinded studies on purified legume components, such as
fiber or protein, are presented in group 4: most of these stud-
ies are on narrow-leaf lupin. An Australian study (Hall et al.,
2005a) investigated the effects of adding lupin kernel fiber to
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TABLE 6
Characteristics of clinical trials on non-soy legume
Reference Type of pulse Preparation
Run-in period
(days) Control diet Daily dose (g/day)
Duration
(days)
Mean
age
(years)
1) Unblind studies on whole legume seeds
Jenkins et al., 1983 C. arietinum, L. culinaris,
P. vulgaris
Cooked/canned >1 year stable
parameters
Legumes replace
starchy foods
140 g 120 48
Anderson et al., 1984 P. vulgaris Cooked 7 Matched American diet 115 g dry weight 21 34-66
Anderson et al., 1990 P. vulgaris Canned 7 Matched American diet 69 g 21 58.0
Cobiac et al., 1990 P. vulgaris Canned 20 Spaghetti 440 g wet weight 28 29-65
Mackay and Ball,
1992
P. vulgaris Cooked 28 (low fat diet) Matched diet 80 g 42 28-66
Duane, 1997 L. culinaris,P. sativum,
P. vulgaris
Cooked no Matched diet 120 g dry weight 42-49 58
Oosthuizen et al.,
2000
P. vulgaris Extruded dry beans
as extruded
products
28 (usual diet) Usual diet 110 g dry weight 28 48.0
Winham andHutchins,
2007
P. vulgaris Canned no 1/2 cup canned carrot 1 half cup 56 45.9
Pittaway et al., 2008 C. arietinum Cooked 28 Usual diet 182 g, 4 times per
week
120 52.2
Nouran et al., 2009 A. hypogea Roasted and salted no Usual diet 60–93 g per day
(=20% daily
energy)
28 25-65
Hermsdorff et al.,
2011
C. arietinum,L. culinaris,
P. sativum
Cooked,
hypocaloric diet
no Usual food 160–235 g,
4 times per week
56 36
Abeysekara et al.,
2012
C. arietinum, L. culinaris,
P. sativum, P. vulgaris
Cooked (many
kinds of foods)
no Usual food 150 g dry weight 60 +60 59.7
2) Unblind study on model food from whole seed
Nowicka et al., 2006 L. albus Lupin drink (6.7%
protein), lipid diet
>3 months stable
parameters
Lupin drink replaces
cow milk in a low
lipid diet
500 mL 90 49.7
154
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3) Blind studies on model foods containing whole kernel flour
Fruhbeck et al., 1997 V. faba∗Raw flour no Powder for smashed
potato +
lactalbumin
90 g 30 19.6
Fruhbeck et al., 1997 V. faba∗Raw flour no Powder for smashed
potato +
lactalbumin
90 g 30 19.8
Belski et al., 2011 L. angustifolius bread, biscuits,
pasta (25-40%
lupin flour),
hypocaloric diet
no Usual food items Substitution of
normal foods
365 46.5
4) Blind studies on purified fiber or proteins
Hall et al., 2005 L. angustifolius 50% soluble +
50% insoluble
no Matched with the
exclusion of fiber
25 g fiber 28 41.0
Weisse et al., 2010 L. angustifolius Dietary bar (protein
isolate NL-LUP)
no Casein bars 35 g protein 42 43.9
Sirtori et al., 2012 L. angustifolius Dietary bar (protein
isolate NL-LUP)
28 day, low
lipid diet
Casein bars Two bars/day
(35 g protein)
28 52.3
Sirtori et al., 2012 P. sativum Dietary bar (protein
isolate - pisane)
28 day, low
lipid diet
Casein bars Two bars/day
(35 g protein)
28 52.5
B¨
ahr et al., 2013 L. angustifolius Drink based on
protein isolate
NL-LUP
no Milk protein isolate 500 mL (25 g
protein)
28 49.5
∗The paper reports separate data for hypercholesterolemic subjects (see Table 7).
155
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TABLE 7
Lipid responses in clinical studies on non-soy legumes
Total Cholesterol LDL-C HDL-C
Total cholesterol /
HDL-C LDL-C / HDL-C TAG
Reference
Number of
patients Gender Design
Control
value
(mg/dL)
Change
(mg/dL)
Control
value
(mg/dL)
Change
(mg/dL)
Control
value
(mg/dL)
Change
(mg/dL)
Control
value Change
Control
value Change
Control
value
(mg/dL)
Change
(mg/dL)
1) Unblind studies on whole legume seeds
Jenkins et al., 1983 7 M uncontrolled 269.0−20∗188.6−12.437.4+2.6 238 −69∗
Anderson et al., 1984 10 M PARA 300 −56∗221 −51∗32.3−4.1 233 −8
Anderson et al., 1990 24 M PARA 294.6−31.3∗200.4−17.5∗41.70.0 256 −39∗
Cobiac et al., 1990 20 M CROSS 246.3−1.2 181.1−0.848.3−1.543+5
Mackay and Ball,
1992
39 M, F CROSS 266.8−1.9 161.80 44.4+4.73.76 −0.25∗59 −1
Duane, 1997 9 M CROSS 204 −10∗138 −12∗32 −1 172 +12
Oosthuizen et al.,
2000
22 M CROSS 234.7+3.00 182.1+14.70.97 −5.06.26 +1.06 4.86 +1.2 145 +10
Winham and
Hutchins, 2007
23 M, F CROSS 216 −13∗135 −8∗58 −24.0−0.2∗139 −17
Pittaway et al., 2008 45 M, F CROSS 254.0−7.7∗nr −7.3∗nr nr nr nr
Nouran et al., 2009 54 M CROSS 255.0+10.1 170.0+7.033.56.1∗7.8−1.0∗5.2−0.7∗232 +1.8
Hermsdorff et al.,
2011
15 M, F PARA 215.0−25∗142.2−14∗49 0 99 −2
Abeysekara et al.,
2012
87 M, F CROSS 176.7−17.8∗113.3−14.745.6−4.681+8
2) Unblind study on model food from whole seed
Nowicka et al., 2006 42 M, F
smokers
uncontrolled 239.9−16.4∗154.1−12.3∗58.8−2.7 137.1 −6.2
3) Blind studies on model foods containing whole kernel flour
Fruhbeck et al., 1997 10 M PARA 206.5−5.1∗141.5−6.9∗37.1+6.6∗5.59 −0.96∗3.84 −0.75∗139 −22∗
Fruhbeck et al., 1997 10 M PARA 240.9−16.6∗169.0−12.4∗40.6+6.2∗5.96 −1.13∗4.19 −0.81∗158 −51∗
Belski et al., 2011 55 M, F PARA,
DB
200.3−3.9 127.2−3.551.40.43.9−0.12.5−0.1 85 +7
4) Blind studies on purified fibre or proteins
Hall et al., 2005 38 M CROSS,
SB
207.6−8.4∗133.8−7.348.7+0.64.45 −0.17∗2.89 −0.15∗134 −3
Weisse et al., 2010 22 M, F PARA,
DB
218.8−19.2139.6−12.067.714.72.24 −0.11 110 +7
Sirtori et al., 2012 25 M, F PARA,
DB
274.0−11.6∗188.2−5.656.0−1.3 145.6 −19.1
Sirtori et al., 2012 25 M, F PARA,
DB
271.1−2.7 182.47.256.9+1.5 149.8 −18.2
B¨
ahr et al., 2013 33 M, F CROSS,
DB
253.2−2.0 170.0−3.157−2.03.32 +0.02 157 +3
PARA =parallel design; CROSS =cross over design; SB =single blind; DB =double blind; nr =not reported. Significant changes vs. initial values are in bold, when they are
significant also vs. the control they are labelled with an asterisk (∗).
156
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GRAIN LEGUMES 157
breads, muffins, and other foods: small but significant decreases
of total and LDL-C were observed, although most of the sub-
jects were normolipidemic. A German study (Weisse et al.,
2010) considered dietary bars containing either the lupin pro-
tein isolate NL-LUP or casein (control bar). The lupin bar sig-
nificantly decreased the lipid parameters compared to baseline,
which, however, were improved also after the casein diet. It is
important to underline that the authors did not indicate the pres-
ence of a run-in period aimed to stabilise the lipid parameters
before starting the dietary intervention. In our experience, this is
crucially important for obtaining constant lipid parameters in the
control group and reliable measures of change (or non-change)
in the treatment group. Another study performed in Germany has
investigated the effect of 25 g NL-LUP lupin protein incorpo-
rated into a lupin drink: this lupin protein intake did not change
the lipid profile in statistically significant way (B¨
ahr et al., 2013).
The final study (Sirtori et al., 2012b) was performed in Italy
and was aimed at evaluating the effect of narrow-leaf lupin pro-
tein (NL-LUP) or a commercial pea protein isolate and their
combinations with soluble fibers (oat fiber or apple pectin) on
plasma total and LDL-C levels. After a four-week run-in pe-
riod, participants were randomized into seven treatment groups,
each consisting of 25 participants. Each group consumed two
bars containing specific protein/fiber combinations. The refer-
ence group consumed casein +cellulose; the second and third
groups consumed lupin or pea proteins +cellulose; the fourth
and fifth groups consumed casein +oat fiber or apple pectin;
the sixth group and seventh group received bars containing com-
binations of pea protein and oat fiber or apple pectin, respec-
tively. For simplicity, only the data of the lupin protein +cel-
lulose and pea protein +cellulose arms are reported in Table 6
and 7. Bars containing lupin protein +cellulose (−116 mg/L,
−4·2%), casein +apple pectin (−152 mg/L,−5·3%), pea pro-
tein +oat fiber (−135 mg/L, −4·7%) or pea protein +apple
pectin (−168 mg/L, −6·4%) resulted in significant reductions of
total cholesterol levels, whereas no plasma cholesterol changes
were observed in the subjects consuming the bars containing ca-
sein +cellulose, casein +oat fiber or pea protein +cellulose.
These findings indicated that lupin protein is more effective than
pea protein in lowering cholesterol and that the combination of
soluble fiber with plant protein may have interesting applica-
tions in this area.
The Anderson meta-analysis on soybean (Anderson et al.,
1995) demonstrated that the main significant predictor of the
change of cholesterol concentration is the square of initial serum
cholesterol. It is therefore useful to compare (Figure 1) the net
changes in total cholesterol detected in the studies on non-soy
legumes (white circles) as a function of baseline total choles-
terol levels with those observed in studies on soybean (black
triangles). Apparently, the studies on non-soy legumes seem to
belong to the same population of the studies on soybean.
The same meta-analysis on soybean (Anderson et al., 1995)
suggested dividing the studies in quartiles as shown in Ta-
ble 1. Classifying with the same criteria the studies on non-soy
FIG. 1. Comparison of the effects of consuming soybean (Sirtori et al., 2007)
or on-soy legumes (Table 7): changes in total cholesterol as a function of baseline
cholesterol in available studies.
legumes (single arms), two belong to the first quartile, thirteen
to the second quartile, and six to the third quartile. Not surpris-
ingly, no study belongs to the fourth quartile, since treatment
with statins or other lipid-lowering drugs is now mandatory in
these cases. The subjects of Q1 had mean reductions of the total
cholesterol concentration of 10.6 mg/dL; those of Q2 had mean
reductions of 19.8 mg/dL; those of Q3 had a mean reduction
of 20.6 mg/dL. These values are in reasonable agreement with
those reported in Table 1. This analysis clearly indicates that
not only soybean, but also non-soy legumes may be useful in
the prevention of hypercholesterolemia providing wide possi-
bilities of making this kind of dietary regimen more palatable
and acceptable.
C. Mechanism of the Hypocholesterolemic Activity
At the transcriptional level, cholesterol biosynthesis and up-
take are closely regulated through a negative feedback control.
When there is an accumulation of cholesterol into the cell, the
endogenous cholesterol biosynthesis mediated by 3-hydroxy-3-
methylglutaryl coenzyme A (HMGCoA) reductase is reduced
and the number of LDL receptors decreases. Conversely, when
there is a depletion of intracellular cholesterol, the activity of
HMGCoA reductase is elevated and there are more numerous
LDL receptors on the cellular membrane.
The sterol regulatory element binding proteins (SREPBs) are
a family of transcription factors that regulates the gene expres-
sion of most of the enzymes involved in cholesterol biosynthe-
sis. The SREBP family includes SREBP-1a, SREBP-1c, and
SREBP-2. SREBP-2 is directly involved in the regulation of
cholesterol metabolism, whereas SREBP-1a and SREBP-1c are
involved in the fatty acid and TAG metabolism. Usually both
SREBP-1 and SREBP-2 are associated with another endoplas-
mic reticulum membrane protein, the SREBP-cleavage activat-
ing protein (SCAP). This complex behaves in two different ways
depending on the intracellular sterols levels. When there is a de-
crease or a depletion of intracellular sterol, the SREBP/SCAP
complex moves from the endoplasmic reticulum to the Golgi
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158 A. ARNOLDI ET AL.
apparatus. When the complex arrives into the Golgi apparatus,
SREBP is cleaved by two proteases and the transcription factor
domain is activated. In contrast, when the level of intracellu-
lar sterol is high, SCAP interacts with Insigs. This interaction
hinders the movement of the SREBP/SCAP complex from the
endoplasmic reticulum to the Golgi apparatus (Goldstein et al.,
2006; Sato, 2010).
Although numerous studies have shown that grain legumes
may play an important role in the prevention of hypercholes-
terolemia, the molecular mechanisms of this activity is not
clearly understood yet: some incomplete information is now
provided from studies on soybean.
1. Soybean
The success of the clinical studies on the cholesterol-
lowering activity of soybean protein has stimulated numerous
investigations aimed to assess: i) the bioactive components in
soybean, and ii) the mechanism of action at a molecular level.
Some studies, both on animal models and in humans, have
investigated whether the hypocholesterolemic effects of soy is
linked to the activation/depression of the LDL-receptors. A
study has shown that soy protein is able to reverse the dra-
matic down-regulation of liver LDL-receptors observed in rats
on cholesterol/cholic acid dietary regimens vs. casein (Lovati
et al., 2000; Sirtori et al., 1984). A clinical trial (Lovati et al.,
1987) investigated the expression of LDL-receptor in patients
affected by familial hypercholesterolemia, treated with animal
proteins or with soy protein with cholesterol added to balance
the two diets. In this study, the LDL degradation was moni-
tored in circulating lympho-monocytes (used as mirror images
of hepatocytes). The soy protein diet determined a marked LDL-
C reduction as well as an 8-fold increase of LDL degradation,
whereas there were minimal changes in LDL-C levels or LDL-
receptor activity after the animal protein diet. The findings of
this study, clearly suggesting that some soy protein components
are able to up-regulate the LDL-receptor mediated LDL degra-
dation, were subsequently confirmed in individuals with lesser
cholesterol elevations (Baum et al., 1998).
Studies in monkeys (Anthony et al., 1996) and the Ander-
son meta-analysis (Anderson et al., 1995) suggested that soy
isoflavones, i.e. genistein and daidzein and their glycosides, may
possibly play a role in cholesterol reduction. This is, however,
contradicted by the fact that numerous effective studies included
in the same meta-analysis were carried out with soy concentrates
or isolates containing less than 30–40 μg/kg isoflavones (Sirtori
et al., 1995; Sirtori et al., 1997) and that pure genistein does not
modulate the up-take and degradation of the LDL-receptor in
HepG2 cells (Lovati et al., 2000).
The controversial role of isoflavones was clarified by a study
in ovariectomised female monkeys, which showed that the ad-
dition of a semi purified soy isoflavone-rich ethanol extract to
a casein diet failed to improve cholesterolemia vs. native soy
protein (Greaves et al., 1999). The same authors, subsequently,
confirmed that a soy protein diet reduces cholesterolemia in
ovariectomised adult female monkeys, also by partially inhibit-
ing cholesterol absorption, whereas, a semi-purified soy extract,
rich in isoflavones, added to casein did not exert any lipid low-
ering effect (Greaves et al., 2000).
In general, isoflavone-free soybean protein is prepared by
extraction with hot ethanol. Since this drastic treatment might
change the protein structure, an innovative investigation pro-
posed instead to use column chromatography for preparation of
the isoflavone-free protein (Fukui et al., 2002). The results of
this study, reported in Table 5, clearly indicate that the activities
of the native soybean protein and the isoflavone-free sample are
equivalent.
A detailed discussion on the role of isoflavones in the hypoc-
holesterolemic activity of soy protein may be found in a recent
review (Sirtori et al., 2005). Other studies have addressed the
possible role of soy proteins per se in the reduction of choles-
terolemia. The two major proteins in soybean are legumins and
vicilins (Gianazza et al., 2003). The latter’s consist of different
combinations of three major subunits, α,α, and β.
One of the first studies aimed at understanding the hypoc-
holesterolemic effect of soy protein at the molecular level was
performed with the hepatoma cell line (HepG2), which is highly
sensitive to factors regulating LDL receptor expression and
cholesterol biosynthesis / breakdown. The experiment consisted
of tracking the uptake and degradation of labelled LDL, in order
to identify the soy protein component/s potentially responsible
for the cholesterol lowering effect (Lovati et al., 1992). The
vicilins appeared to be primary responsible for the hypocholes-
terolemic effect of soy protein, since they were able to stimulate
both the uptake and degradation of 125I-labeled LDL in HepG2
cells, whereas the legumins appeared to be inactive.
After this crucial experiment, many efforts were aimed at
identifying the vicilin subunit responsible for the hypocholes-
terolemic effect. One investigation examined the effect of the
vicilin subunits vs. the whole vicilins (Lovati et al., 1998). It
was found that the incubation of HepG2 cells with the purified
α+αsubunit fraction increased the uptake and degradation of
labelled LDL, whereas the βsubunit was ineffective.
In a following study, it was possible to directly test the α
subunit (Manzoni et al., 2003): the up-regulation of the LDL
receptor by the αsubunit was significantly greater than that
of control cells. This study also revealed a potentially interest-
ing association of soybean vicilins with other proteins, such as
thioredoxin-1 and cyclophilin-B, both involved in cell protec-
tion against oxidative stress.
Finally, in the hypocholesterolemic rat model, at 20 mg/kg
body weight, the αsubunit significantly decreased plasma
cholesterol and triglyceride (Table 5) and also normalized the
β-VLDL receptor activity (Duranti et al., 2004). Similar ac-
tivities were achieved feeding the animals the purified vicilins
at 200 mg/kg body weight. These data are crucially important,
since they represent the first direct in vivo confirmation that the
αsubunit is responsible for the hypocholesterolemic activity of
soy protein.
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GRAIN LEGUMES 159
Recently, two studies demonstrated the ability of a truncated
form of the vicilin αsubunit (tα) (Consonni et al., 2010) and
a recombinant polypeptide αE polypeptide (αE) (Consonni
et al., 2011), in modulating cholesterol homeostasis and the
activity of the LDL receptor in HepG2 cells. By monitoring the
uptake and degradation of 125I-LDL, it was demonstrated that
the treatments with tαor αE produced significant increases
in LDL uptake and degradation, similar to those found in cells
incubated with simvastatin (a potent inhibitor of cholesterol
biosynthesis).
Another study investigated the effect of soy vicilins and legu-
mins on serum lipid levels and metabolism in the liver of nor-
mal and genetically obese mice (Moriyama et al., 2004). In this
study, mice fed vicilins had serum TAG, glucose, and insulin
level lower than casein-fed mice. This effect may have been due
to an increase in the mRNA level of acyl-CoA oxidase (fatty
acid β-oxidation related enzyme) and may be correlated to the
decreased mRNA levels of SREBP-1 and SREBP-2 in the liver
of vicilin-fed mice vs. casein-fed mice. In addition, the faecal
excretion of TAG of the vicilin-fed mice was higher than in
casein-fed mice.
An octa-peptide FVVNATSN, derived from the β-subunit of
soy vicilins, was purified, characterized, and shown to be able
to activate the LDL receptor in the HepG2 cells (Cho et al.,
2008). Moreover, short peptide mixtures derived from the vi-
cilin are able to alter lipid metabolism, by decreasing plasma
TAG synthesis and suppressing the secretion into the medium
of apolipoprotein B-100 from HepG2 cells (Mochizuki et al.,
2009). These vicilin-derived peptides modified the gene ex-
pression profile of many proteins known to be involved in the
reduction of hypertriglyceridemia. Another research group has
identified and isolated two hypocholesterolemic peptides, LPYP
and IAVPGEVA, from the legumins after hydrolysis with trypsin
and pepsin, respectively (Pak et al., 2005). Since these peptides
were shown to be able to inhibit the activity of HMGCoA re-
ductase, a relevant enzyme in the cholesterol biosynthesis, they
potentially are good candidates for being responsible of the
hypocholesterolemic activity of soy protein.
2. Pea
Information on the mechanism of lipid modulation action of
the grain legumes is very scarce and not very detailed and only
pea and lupin have been investigated. In a study in the hyperc-
holesterolemic rat model, a dietary treatment with a commercial
pea protein isolate reduced plasma cholesterol and TAG levels
vs. the casein treatment (Rigamonti et al., 2010). This investiga-
tion also demonstrated an up-regulation of the genes involved in
hepatic cholesterol uptake, since the mRNA levels of the LDL-
receptor was higher in pea protein fed rats than in those fed
casein.
In a similar rat model (Spielmann et al., 2008), pea proteins
increased the relative mRNA concentrations of the transcription
factor, SREBP-2, and its target genes, HMG-CoA reductase,
and also enhanced the LDL-receptor and cholesterol 7-alpha-
hydroxylase (CYP7A1). At the same time, the pea protein treat-
ment led to a reduced hepatic cholesterol concentration through
the formation and excretion of bile acids. Thus, the increase in
gene expression of the SREBP-2/LDL-receptor pathway might
be a way to balance the cholesterol loss caused by the bile acid
synthesis.
3. Lupin
Lupin and soybean seeds have some compositional similar-
ities (Table 2), including similarities of their storage protein
sequences (Sirtori et al., 2010b; Wait et al., 2005). However,
they also have some major differences, in particular in their
isoflavone contents, which is 1-2 mg/g in soybean and minimal
in lupin (Sirtori et al., 2004).
Only a few studies, all in animal models, have investigated
the mechanism of the hypocholesterolemic activity of lupin pro-
tein. An investigation showed that a reduced plasma TAG levels
in hypercholesterolemic rats treated with a white lupin protein
isolate was in part due to the down-regulation of the SREBP-1c
mRNA in the liver, which led to a reduction of hepatic fatty
acid synthase (Spielmann et al., 2007). Another study, how-
ever, observed lower hepatic mRNA concentrations of genes
involved in fatty acid synthesis, and a parallel up-regulation of
genes involved in TAG hydrolysis (Bettzieche et al., 2008b). A
third study showed that the lupin-fed rats displayed significantly
higher hepatic mRNA levels of SREBP-2, the major transcrip-
tional regulator of intracellular cholesterol levels, and CYP7A1,
the rate-limiting enzyme in bile acid biosynthesis (Parolini et al.,
2012).
The main storage proteins in lupin are the acidic legumins (α-
conglutin) and vicilins (β-conglutin), as well as small amounts
of γ-conglutin, a basic 7S globulin (Duranti et al., 1981). γ-
Conglutin enhanced the uptake and degradation of LDL-C in
HepG2 cells (Sirtori et al., 2004), but did not modulate the lipid
profile when tested in the hypercholesterolemic rat model (G.
Chiesa, personal communication).
4. Final considerations
Although a number of published studies have already demon-
strated potentiality beneficial effects of pulses on dyslipidemia,
more research is needed in this area to get satisfactory infor-
mation on the real active components in both soy and non-soy
legumes and to achieve a complete picture of their mechanism
of action. Useful mechanistic data may be provided by future
investigations on the effect of legume components on the choles-
terol biosynthetic pathways in cell culture studies.
V. HYPOTENSIVE ACTIVITY OF GRAIN LEGUMES
A main risk factor for cardiovascular disease is hypertension,
defined by the World Health Organization (WHO) as exceeding
90 mmHg for diastolic arterial pressure and 140 mm Hg for
systolic pressure. High blood pressure is usually treated with
drugs, such as angiotensin receptor blockers, beta-blockers, cal-
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160 A. ARNOLDI ET AL.
cium channel blockers, diuretics and angiotensin I converting
enzyme inhibitors (Aronow, 2012). Interestingly, a few experi-
mental and clinical investigations have shown that some plant
proteins may be useful in maintaining a correct blood pressure.
Most of the published research in this area is on lupin.
A. Animal and Clinical Studies
1. Mixed grain legumes
A study (Table 6), aimed at investigating the effect of a
legume-based hypocaloric diet on proinflammatory status and
the metabolic features of overweight/obese subjects, provided
useful data on blood pressure (Hermsdorff et al., 2011). Thirty
obese subjects (17 M/13 F) were randomly assigned to two
groups: control and treatment. The control group followed a
balance diet without legumes, whereas the treatment group con-
sumed a diet including four different servings (160–235 g) per
week of lentils, chickpeas, peas or beans. The legume treatment
improved the lipoprotein profile (Tables 7) and also lowered the
systolic and diastolic blood pressure by 5 mmHg and 2 mmHg,
respectively, vs. the control diet.
2. Lupin
In an uncontrolled study on subjects with moderate hyper-
cholesterolemia and hypertension, 42 smokers consumed 35 g
white lupin protein daily in a model beverage (Tables 6 and
7) (Nowicka et al., 2006). This treatment significantly reduced
the systolic blood pressure by 9.5 mmHg after one month and
9.1 mmHg after three months, whereas it lowered the diastolic
blood pressure by 3.0 mmHg and by 4.4 mmHg, respectively,
with stronger effects in mild hypertensive subjects.
Two long-term randomized controlled studies showed that
the consumption of a lupin flour enriched bread produced very
small statistically significant decreases in blood pressure vs. the
control bread (Belski et al., 2011; Lee et al., 2009). Overweight
and obese subjects (88) consumed white wheat bread (control
group) or a lupin flour-enriched bread (treated group) for 16
weeks. At the end of this period, the differences in the treatment
group vs. the control group were −3.0 mmHg in the systolic
blood pressure, −0.6 mmHg in the diastolic blood pressure, and
−3.5 mmHg in the pulse pressure (Lee et al., 2009).
The effect of lupin-enriched foods (bread, biscuits and pasta)
was evaluated in a double-blind trial during 12 months (Ta-
bles 6 and 7) (Belski et al., 2011). Normotensive participants
(n=131) were randomly assigned to consume lupin-enriched
foods or high carbohydrate control foods. At month 12, the 24-h
ambulatory systolic (-1.3 mmHg) and diastolic (−1.0 mmHg)
blood pressures of the lupin group were significantly lower than
in the control group.
The hypotensive effect of lupin protein was also investigated
in vivo in Goto-Kakizaki rats, which develop hypertension when
fed a high-salt diet (Pilvi et al., 2006). The rats were fed a 6%
NaCl diet containing either lupin or soy protein isolates (20%
weight/weight) for two weeks. At the end of the treatment,
the systolic blood pressure was 18.6 mmHg lower in the lupin
group and 12.0 mmHg lower in the soy group than in the con-
trol group. In addition, both lupin and soy treatments normalised
the decreased vasoconstriction observed in the NaCl-fed con-
trol group, but only the lupin treatment improved the impaired
endothelium-dependent vasodilatation (Pilvi et al., 2006).
B. In Vitro Studies on ACE-Inhibitory Peptides
A possible explanation of the mild hypotensive activity ob-
served in these human and animal studies is that legume proteins
are cleaved in the gastrointestinal apparatus generating bioactive
peptides previously encrypted in the parent protein sequence. In
fact, it is known that some peptides are able to inhibit the an-
giotensin I converting enzyme (ACE, EC 3.4.15.1), which plays
an important role in regulating blood pressure in the renin-
angiotensin system, because it catalyses the conversion of the
biologically inactive angiotensin I to the potent vasoconstrictor
angiotensin II and inactivates the potent vasodilator bradykinin
(Figure 2) (Skeggs et al., 1956; Yang et al., 1970). Inhibitors
bind tightly to the ACE active site competing with angiotensin
I for occupancy; as a consequence, ACE cannot convert an-
giotensin I to angiotensin II (Skeggs et al., 1956). ACE is widely
distributed in mammalian tissues, as membrane bound ectoen-
zymes in vascular and endothelial cells, and in several other cell
bradykinin no acve pepde
ACE-inhibitors
angiotensin I angiotensin II vasoconstricon
ACE
angiotensinogen
FIG. 2. Schematic mechanism of action of angiotensin converting enzyme and ACE-inhibitors.
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GRAIN LEGUMES 161
types, such as absorptive epithelial, neuroepithelial and male
germinal cells (Li et al., 2004). It is a zinc metallopeptidase,
which is activated by chloride and has broad in vitro substrate
specificity (Li et al., 2004).
Many common hypotensive drugs (e.g., captopril, enalapril,
lisinopril) act as ACE inhibitors (Aronow, 2012).
The first ACE-inhibitory peptides from foods were identi-
fied in milk (Nakamura et al., 1995; Vermeirssen et al., 2004a),
but some data are also available on plant proteins. Three re-
views have addressed different aspects of the ACE-inhibitory
activity of plant proteins (Guang and Phillips, 2009; Hernandez-
Ledesma et al., 2011; Roy et al., 2010), but none has considered
thoroughly all legumes. This section discusses a selection of rel-
evant studies in this area, including also soybean for comparison.
Before discussing these data, it is important to observe that
a direct comparison of the experimental results from different
papers is a difficult task. The reason is that the ACE-inhibitory
activity of each sample depends on the protein extraction pro-
cedures, the resistance of the proteins to proteolysis, the treat-
ments before proteolysis, the possible presence of contaminants
and the composition of the hydrolysates, which is related to
the specificity of the hydrolytic enzyme. The reaction parame-
ters (pH, temperature, enzyme/substrate ratio, and contact time
of the hydrolytic reaction) are equally of outmost importance
(Barbana and Boye, 2010).
Different methods for the measurement of the ACE-
inhibitory activity are reported in the literature using various
substrates, medium, and analytical techniques (Cushman and
Cheung, 1971; Lam et al., 2007; Vermeirssen et al., 2002; Wu
et al., 2002). Numerous papers still use the method developed
by Cushman and Cheung in 1971, based on the determination
of the concentration of hippuric acid (HA), which is formed
from hippuryl-histidyl-leucine (HHL), a substrate that shows a
close resemblance to the physiological substrate angiotensin I,
by the action of ACE; HA is extracted with ethyl acetate and its
concentration is determined spectrophotometrically at 228 nm.
Although this assay is simple and cheap, it has some limitations
when it is applied to the complex peptide mixtures derived from
the hydrolysis of plant proteins, in particular due to the pres-
ence of interfering molecules that can alter spectrophotometric
measures. For this reason, in the last years chromatographic
methods based on HPLC coupled with DAD detector were de-
veloped (Lam et al., 2007; Wu et al., 2002).
Finally, another critical point in the comparison of ACE-
inhibition values is the methodology used for assessing the pep-
tide concentration. Many authors report peptide concentration
without clearly indicating how it was determined or do not eval-
uate it. In a recent paper (Boschin et al., 2014) the peptide
concentration was measured according to a literature method
specific for peptides (Goa, 1953; Levashov et al., 2009), based
on chelating the peptide bonds by Cu (II) in alkaline media and
monitoring the change of absorbance at 330 nm vs. a standard.
A comparison among different legumes was performed in a
recent paper considering the ACE-inhibitory activity of peptide
mixtures obtained by digesting the proteins of several legumes
with pepsin (Boschin et al., 2014). All peptide mixtures were
able to inhibit the ACE activity, but chickpea, common bean,
lentil and pea were only moderately active (IC50 values equal to
673, 633, 606, and 595 μg/mL, respectively), whereas soybean,
white and narrow-leaf lupin were the most active, with IC50
values equal to 224, 268, and 226 μg/mL. Of course, it is very
feasible that using different proteolytic enzymes the order of
activity of peptides from different legumes may change.
1. Soybean
Numerous investigations have been published on the ACE-
inhibitory activity of soybean hydrolysates, with IC50 values
ranging from 21 to 1730 μg/mL. The peptide mixtures ob-
tained from the digestion of soy protein with alcalase had an
IC50 value of 340 μg/mL and 65 μg/mL, either after filtration
through a 10,000 Da cut off membrane or fractionation on cation
exchange resin, respectively (Wu and Ding, 2002). In another
investigation, a pepsin/pancreatin treatment produced a peptide
mixture with IC50 value equal to 280 μg/mL (Lo and Li-Chan,
2005); a further chromatographic fractionation resulted in active
fractions with IC50 values ranging from 130 to 930 μg/mL. In
this study, it was shown that the peptides with lower molecular
weight and higher hydrophobicity were the most active.
The purified glycinin fraction of soybean was digested with
three different proteases, protease-P, trypsin and chymotrypsin
(Mallikarjun Gouda et al., 2006). The peptides obtained using
protease-P exhibited the most potent ACE-inhibitory activity
(4.5 μgN
2) followed by those obtained by trypsin hydrolysis
(9.0 μgN
2). In the same paper, a careful fractionation permitted
the isolation and characterization of one active peptide with
the sequence VLIVP with an IC50 value equal to 1.69 μM.
Very recently, papain and pepsin were used for digesting total
soy protein and two purified fractions, i.e. β-conglycinin and
glycinin (Margatan et al., 2013). The papain hydrolysate of the
β-conglycinin fraction was the most active (170 μg/mL).
Finally, two ACE-inhibitory peptides were isolated and char-
acterized from soy hydrolysates and soy fermented food, e.g.
DLP, and DG (Wu and Ding, 2002; Gibbs et al., 2004).
2. Peanut, chickpea, and lentils
A total protein extract from peanut digested with alcalase
gave a peptide mixture with an IC50 value of 134 μg/mL (Guang
and Phillips, 2009). More highly active peptides fractions were
isolated with further filtration and purification steps, finally re-
sulting in a very active peptide KAFR (IC50 =16.9 μg/mL). A
comparison between pepsin/pancreatin and alcalase treatment
showed that the latter results in peptides mixtures with the high-
est ACE-inhibitory activity (Quist et al., 2009). Different frac-
tions were obtained by RP-HPLC for both enzymatic systems
and for raw and toasted peanuts. The alcalase digestion of raw
peanut proteins exhibited IC50 values of 8.7-122 mg/mL, and
those from roasted flour of 12-235 mg/mL; IC50 values of 7.9-
65.9 mg/mL, and 11-36 mg/mL for raw and roasted peanut,
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162 A. ARNOLDI ET AL.
from the pepsin/pancreatin system were respectively observed
(Quist et al., 2009).
Chickpea proteins digested with alcalase gave an IC50 of
190 μg/mL (Pedroche et al., 2002). Two different varieties
of chickpea produced ACE-inhibitory activity peptide mixtures
with IC50 values equivalent to 229 and 140 μg/mL, when di-
gested with pepsin/trypsin/α-chymotrypsin; equivalent to 316
and 228 μg/mL when digested with alcalase/flavourzyme; and
equivalent to 282 and 180 μg/mL when digested with papain
(Barbana and Boye, 2010).
The treatment with alcalase of the legumin fraction of chick-
pea produced a hydrolysate with an IC50 of 180 μg/mL, whereas
fractionation of the hydrolysate by reverse phase chromatogra-
phy yielded six highly active peptides with IC50 values ranging
from 11 to 21 μg/mL (Yust et al., 2003).
A recent paper has evaluated the ACE-inhibitory activity of
alcalase, papain, and pancreatin hydrolysates from fresh and
hardened chickpea (Medina-Godoy et al., 2012). The latter is
a hard-to-cook sample obtained when grains were stored un-
der adverse conditions of high temperature and high relative
humidity. Hydrolysates from hardened grains showed higher
antihypertensive potential than those from the fresh ones. This
difference in activity may be a result of the protein changes
occurring during the hardening process (e.g. free radical oxida-
tion) making the protein from hardened grain more susceptible
to protease cleavage. Moreover, after separation of the main
protein fractions, it was demonstrated that the peptides derived
from the globulins were more active than those deriving from
the albumins (Medina-Godoy et al., 2012).
In the case of lentil, IC50 values reported in literature are
spread in a very wide range from 8 to 890 μg/mL depend-
ing on the sample (green or red lentils), enzyme used, thermal
treatment applied before the digestion, and protein fractiona-
tion (Akillioglu and Karakaya, 2009; Barbana and Boye, 2011;
Boye et al., 2010; Roy et al., 2010). Differences between the
activity of the peptides mixtures obtained from red and green
lentils were observed after treating with a multi-enzymatic sys-
tem (pepsin/trypsin/α-chymotrypsin), aimed at mimicking the
gastrointestinal system (GIS), or after digestion with papain
(P), or alcalase/flavourzyme (AF), or bromelain (B) (Barbana
and Boye, 2011). For red lentils IC50 values of 90, 86, 154, and
190 μg/mL, and for green lentils of 53, 80, 152, and 174 μg/mL,
were obtained for GIS, P, AF, and B, respectively.
In another report, purified protein fractions, i.e., albumins,
legumins, and vicilins, were investigated after digestion with
trypsin, obtaining IC50 values of 476, 509, 539 μg/mL re-
spectively, whereas the IC50 of the total protein extract was
440 μg/mL (Boye et al., 2010).
3. Common bean, Lima bean, cowpea, and mung bean
A specific characteristic of common bean (P. vulgaris)isthe
presence of numerous varieties with very variable appearance
and protein composition, and with local names that change in the
different countries and are difficult to translate. Since only a few
publications indicate the variety, it is very difficult to get a clear
overview of the ACE-inhibitory activity of this species. Diges-
tion with pepsin of common bean cv. Dermason gave peptides
with IC50 values in the range 770-830 μg protein/mL, whereas
the IC50 in the case of cv. Pinto Bean peptides were in the range
150–690 μg protein/mL (Akillioglu and Karakaya, 2009). Navy
bean, black bean, and small red beans are quality groups within
common bean. They were submitted to a two-step digestion
processes using alcalase/flavourzyme (AF) and alcalase/papain
(AP) after a heat treatment (Rui et al., 2012). The best ACE-
inhibitory activity was achieved with AP digestion; with IC50
values for navy bean, black, and small red beans being 68, 83,
and 78 μg/mL, respectively. In a recent report, the same group
purified and characterized several ACE-inhibitory peptides from
red bean proteins hydrolysed with alcalase and papain (Rui et al.,
2013). Protein concentrates of Jamapa bean (a P. vulgaris cul-
tivar) were digested with alcalase and flavourzyme; the best
IC50 values were 61 and 127 μg/mL, respectively (Torruco-Uco
et al., 2009). Proteins of azufrado (sulphur yellow) beans gave
surprisingly low values of IC50 values when digested with al-
calase, thermolysin, and pancreatin (Valdez-Ortiz et al., 2012).
Lima bean proteins were digested with alcalase and
flavourzyme considering different times of hydrolysis; the
best IC50 values were 56 and 6.9 μg/mL, respectively, af-
ter 90 min (Torruco-Uco et al., 2009). In another study, the
pepsin/pancreatin digestion of Lima bean proteins gave pep-
tides with IC50 values in the range 250-690 μg/mL, whereas
with alcalase digestion the IC50 values were in the range 610-
2400 μg/mL, depending on the enzyme/substrate ratio, hydroly-
sis time, and germination of seeds (Chel-Guerrero et al., 2012).
Cowpea proteins were digested with several enzymes
giving peptides mixtures with IC50 values in the range 0.04-
170.6 μg/mL with flavourzyme, 24.3–123 μg/mL with alcalase,
and 44.7–112 μg/mL with pepsin/pancreatin, depending on the
molecular weight of peptide fraction (Segura Campos et al.,
2010).
Mung bean protein isolates hydrolysed with alcalase gave
peptide mixtures with IC50 value equal to 0.63 μg/mL (Li
et al., 2005a; Li et al., 2005b); whereas neutrase was less
effective (Li et al., 2006a). The same Authors identified several
ACE-inhibitory peptides from alcalase hydrolysates: KDYRL,
VTPALR, and KLPAGTLF, having IC50 values equal to 26.5,
82.4, and 13.4 μM, respectively, were the most active (Li et al.,
2006b).
4. Pea
Several published reports are available on pea. In one study
pepsin digestion generates an ACE-inhibitory peptide mixture
with IC50 equal to 117 μg/mL (Vermeirssen et al., 2004b). A
second digestion step performed with trypsin/α-chymotrypsin
in order to simulate a gastrointestinal digestion improved the
IC50 to 76 μg/mL. In another paper, pea proteins digested
with pepsin gave an IC50 of 80 μg/mL, whereas the digestion
with pepsin/trypsin/α-chymotrypsin gave an IC50 of 69 μg/mL
(Aluko, 2008). By ultrafiltration and HPLC separation, several
fractions with very high ACE-inhibitory activity were isolated.
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GRAIN LEGUMES 163
A comparison of several enzymes for digestion of pea pro-
teins showed that papain treatment resulted in more active
ACE-inhibitory peptide mixtures than alcalase, trypsin, chy-
motrypsin, and flavourzyme (Humiski and Aluko, 2007). A gas-
trointestinal simulation of digestion of pea protein performed
with pepsin/trypsin/α-chymotrypsin produced active peptide
mixtures with a IC50 value of 159 μg/mL, while papain was
more effective, giving an IC50 value of 128 μg/mL (Barbana
and Boye, 2010). The peptide mixtures obtained by digesting
pea proteins with alcalase were fractionated on a cationic solid-
phase extraction column and some active peptides were isolated:
IR (IC50 =340 μg/mL); KR (IC50 =340 μg/mL); EF (IC50 =
340 μg/mL) (Li and Aluko, 2010).
5. Lupin
The ACE-inhibitory activities of the peptides obtained by
hydrolysing lupin protein have only recently been investigated
(Boschin et al., 2014). The proteins of the species white and
narrow-leaf lupin,digested with pepsin, gave IC50 values equal
to 268, and 226 μg/mL, respectively. In the same paper (Boschin
et al., 2014), three protein fractions were purified, hydrolysed
and analysed for their ACE-inhibitory activity: a mixture of α+
βconglutin; γconglutin; and δconglutin. The active component
was the α+βconglutin mixture, which was more active than
the total lupin protein extract (IC50 value equal to 138 μg/mL
vs. 268 μg/mL), whereas γconglutin and δconglutin were
inactive. Additionally, this paper demonstrated that the peptides
obtained from the industrial isolate W-LUP containing α+β
conglutins keeps the ACE-inhibitory activity, having an IC50
value of 165 μg/mL. This means that the heat treatment and
the spray-drying process applied for the production of W-LUP
(D’Agostina et al., 2006) did not impair the activity of this
product.
6. Final considerations
The data reported in this section indicate that the presence of
ACE-inhibitory peptides encrypted in the sequence of legume
proteins is a rather common phenomenon. It is thus feasible
that, at least in part, these peptides may be responsible for the
hypotensive activity observed in the few clinical studies avail-
able in the literature (section V.A). Of course, after release from
the parent protein by digestion, the integrity, absorption, and
bioavailability of these peptides are requirements in order to
express their activity in vivo (Hernandez-Ledesma et al., 2011;
Roberts et al., 1999; Vermeirssen et al., 2004a).
A recent review discussed the current literature on ACE-
inhibitory peptides from different foods focusing on the main
methodologies for their production, bioavailability, physiolog-
ical effects in vitro and in vivo, and the structure/activity re-
lationship (Hernandez-Ledesma et al., 2011). The amino acid
sequence and the length of the peptide chain are very crucial for
expressing antihypertensive effects. The preferred C-terminal
amino acids for a strong ACE-inhibitory activity are proline,
lysine, and arginine (Erdmann et al., 2008) and the best length
is 2–5 amino acids, since in this case they are readily absorbed
in the hematic flux. On the contrary, longer peptides (10-51
amino acids), although absorbed, do not function as ACE in-
hibitors, because they are too long to interact with the receptor
site (Erdmann et al., 2008; Roberts et al., 1999).
VI. CONCLUSIONS
The body of research described in this review, clearly indi-
cates that legumes may have a major role in the dietary preven-
tion of hyperlipidemia and hypertension. It would thus be very
advisable to increase their consumption in the daily diet. To
achieve this objective, it would also be very important to have
the easy access to different food items based on legumes, in-
cluding products similar to those currently available made from
soy as well as novel pulse-based products.
ACKNOWLEDGMENT
The authors thank Prof. Paolo Magni (University of Milan,
Italy) and Prof. Stuart Johnson (Curtin University, Australia) for
revision of the manuscript.
FUNDING
The authors are indebted to the European Union Seventh
Framework Programme (FP7/2007-2013), under grant agree-
ment n. 285819, for a post-doc fellowship to C.L. and to the
Italian MIUR, DM n. 593/2000, ART 11 Project N. 13/6, for a
post-doc fellowship to C.Z.
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