Plant Foods Hum Nutr

DOI 10.1007/s11130-013-0353-1

ORIGINAL PAPER

Cooked Common Beans (Phaseolus vulgaris) Protect

Against β-cell Damage in Streptozotocin-Induced
Diabetic Rats
Diego Hernández-Saavedra & Magdalena Mendoza-Sánchez &
Hebert L. Hernández-Montiel & Horacio S. Guzmán-Maldonado &
Guadalupe F. Loarca-Piña & Luis M. Salgado & Rosalía Reynoso-Camacho

# Springer Science+Business Media New York 2013

Abstract Diabetes is a disease characterized by a hypergly- (56.1 %). The preventive-treatment group did not inhibit the
cemic stage that leads to a chronic inflammatory state. We increase of TNF-α and IL-1β, whereas the treatment group
evaluated the in vivo effect of a diet supplemented with 25 % did, compared to the diabetic control. Therefore, N8025 bean
cooked black bean cultivar Negro 8025 (N8025) flour in supplementation can be recommended to control diabetes.
streptozotocin-induced diabetic rats. The effect was assessed
before (preventive-treatment) and after (treatment) the onset Keywords Bean . Phenolic compounds . Diabetes .
of diabetes. There is a significant decrease of total phenolic, Pancreatic damage . Cytokines
tannins and anthocyanins content after cooking, and the con-
centration of most of the single phenols analyzed are only Abbreviations
slightly decreased. The treatment group showed a significant HDL High density lipoprotein
reduction of glucose (22.8 %), triglycerides (21.9 %), total IL-1β Interleukin-1β
cholesterol (29.9 %) and LDL (56.1 %) that correlates with a LDL Low density lipoprotein
protection of pancreatic ß-cells. The diet with N8025 flour N8025 Black bean 8025
before the induction of diabetes did not exert a protective ROS Reactive oxygen species
effect (glucose levels are similar to the diabetic control) but STZ Streptozotocin
they have low levels of total cholesterol (47.5 %) and LDL TA Total anthocyanins
TC Total cholesterol
Electronic supplementary material The online version of this article TAG Triglycerides
(doi:10.1007/s11130-013-0353-1) contains supplementary material, TNF-α Tumor necrosis factor–α
which is available to authorized users.
TPC Total phenolic content
D. Hernández-Saavedra : M. Mendoza-Sánchez : TT Total tannins
G. F. Loarca-Piña : R. Reynoso-Camacho (*)
Research and Graduate Studies in Food Science, School of
Chemistry, UAQ, Santiago de Querétaro, Querétaro, México
e-mail: rrcamachomx@yahoo.com.mx
Introduction
H. L. Hernández-Montiel
Department of Biomedical Research, School of Medicine, UAQ, Diabetes is a progressive disease characterized by high blood
Santiago de Querétaro, Querétaro, México
glucose levels that leads to an increase of metabolic substrate
H. S. Guzmán-Maldonado flux through the mitochondria resulting in reactive oxygen
Campo Experimental Bajío (CEBAJ-INIFAP), Celaya, México species (ROS) overproduction [1, 2]. β-cells are especially
sensitive to excessive ROS due to their low expression of
L. M. Salgado (*)
antioxidant enzymes compared to other cell types. This may
CICATA, Instituto Politecnico Nacional, Santiago de Querétaro,
Querétaro, México explain the higher susceptibility of pancreatic tissue to oxidative
e-mail: lmsalgador@ipn.mx damage [3]. Inflammation and oxidative stress are associated

with other diabetic complications such as kidney or retina
damage [4]. Certain cytokines, such as tumor necrosis
factor-α (TNF-α) and interleukin-1β (IL-1β), are inducers
of β-cell damage in type I diabetes and can be used as early
indicators of autoimmune aggression [5].
Diet plays an important role in diabetic development, and
the consumption of foods with known nutraceutical properties
may help to control it. Common bean (Phaseolus vulgaris) is
a legume regularly consumed in Mexico. It has been used to
improve the diabetic condition, due to its low glycemic index
and phytochemical content [6]. It has been reported that oral
administration of an aqueous or ethanolic extract of
P. vulgaris pods has hypoglycemic and hypolipidemic effects
in which the aqueous extract increases insulin levels and
decreases the activity of gluconeogenic enzymes in the liver.
They have been shown to increase the levels of antioxidant
enzymes [7, 8].
Beans contain a wide variety of chemical compounds with
reported health benefits. Among these are saponins, oligosac-
charides, phytates, and phenols [9]. However, heating damages
these compounds, although, to be eaten, beans must be cooked.
To our knowledge, there are no reports on the possible benefits
of cooked beans on diabetes, although it has been published
that the level of phenols decreases by about 80 % after cooking
and have lower in vitro antioxidant activity [10–12]. The
present study has evaluated the effect of cooked bean con-
sumption on the prevention of pancreatic β-cell damage.
Materials and Methods
Plant Material and Flour Preparation
Phaseolus vulgaris L. was obtained from INIFAP Bajío
(harvest 2008). The beans, cultivar Negro 8025 (N8025),
were washed and cooked (2 h) in a 1:1.6 (w/v) at 96 °C.
They were filtered, dried, and milled. The resulting flour
was preserved by refrigeration.
Extraction and Quantification of Phenolic Compounds
Methanolic extracts were obtained from 1 g of flour mixed
with 10 ml of methanol and shaken for 24 h at RT. After
centrifugation, the total phenolic content was determined by
the Folin-Ciocalteu method [13], the concentration is
expressed as mg of gallic acid equivalents per gram of dry
bean. Condensed tannins were assayed by the vainillin-HCl
method from the methanol solution [14]; the concentration
is expressed as mg of (+)-catechin equivalents per gram of
dry bean. Anthocyanin content was determined from the
methanolic extract using a differential pH method [15], the
values are expressed as milligrams of cyanidin-3-glucoside
equivalents per gram of dry bean using an extinction
Plant Foods Hum Nutr
coefficient of 26,900 L cm−1 mol−1 and a molecular weight
of 449.2 g/mol.
Individual Phenolics (HPLC-DAD) Chromatography
Methanolic extract was used to separate and quantify indi-
vidual phenolic compounds using an Agilent 1100 model
HPLC [16] (Online resource 1).
Animals and Protocol
56 male Wistar rats (250–300 g) were purchased from
Rismart Laboratories (Mexico). Rats were properly housed,
handled daily, and kept at a controlled standard temperature
(20–25 °C) and a 12-h/12-h light/dark cycle. The healthy
control (n=16) and diabetic control groups (n=24) had
unrestricted access to water and were fed ad libitum
(Zeigler rat chow) for five weeks. Animals corresponding
to the preventive-treatment group (n=16) were fed a diet
supplemented with N8025 at 25 % (w/w) throughout the
experiment. At the fourth week of the experiment, diabetes
was induced in the diabetic control, the preventive, and
treatment groups by a single intraperitoneal injection of
streptozotocin (STZ, 45 mg/kg body weight in a 0.01 M
sodium citrate buffer pH 4.5). Five days later, blood glu-
cose levels were determined by a glucose oxidase assay as
measured by a reflective glucometer (Glucose Accutrend,
Roche, Germany). One week after diabetes induction the
animals corresponding to the diabetic control (n=24) were
separated into a diabetic control group (n=16) and a treatment
group (n=8). Half of the animals from the healthy (n=8) and
diabetic control groups (n=8), as well as half of the animals
from the preventive-treatment group (n=8) were sacrificed for
biochemical and histological analyses (Phase 1). One week
after diabetes induction, the treatment group was fed a diet
supplemented with N8025 at 25 % (w/w) until the end of the
experiment. The preventive treatment group continued with
its N8025 supplemented diet. The remaining animals from
the healthy control (n=8), and the diabetic control (n=8)
were fed with normal diet. After four weeks, all animals were
sacrificed and blood samples were immediately collected (Phase
2). The pancreas was immediately removed, cross-sectioned,
and cryopreserved. The tissue was fixed and processed for
paraffin embedding, 3 μm thick sections were stained with
haematoxylin and eosin for routine histopathological diagnosis.
Experiments were performed in accordance with the Animal
Care and Use Protocol of the University of Queretaro (UAQ),
as outlined by the NIH, Publications No. 80–23.
Glycemia, Lipid Profile, and Inflammatory Markers
Total cholesterol (TC), low density lipoprotein cholesterol
(LDL), high density lipoprotein cholesterol (HDL), and
Plant Foods Hum Nutr
triglycerides (TAG) were determined from serum samples
under fasting conditions using commercially available kits
(Randox Laboratories Ltd., UK). Serum levels of interleukin-
1β (IL-1β) and tumor necrosis factor-α (TNF-α) were
quantified using a rat ELISA kit (Invitrogen, USA).
Statistical Analysis
Data are expressed as mean values ± one standard error
(SE). The statistical significance of differences (P<0.05)
between the mean values for the treatment groups was
analyzed by Tukey’s test and a one-way analysis of variance
(ANOVA) using the statistical software package JMP 5.0.1.
Results and Discussion
Functional Characterization
Among the different compounds detected in beans are those
with antioxidant capacity, like phenols, thought to have a
greater effect against hyperglycemia. The cooking process
changes the nutraceutical properties of the food, and hence
we have quantified the amount of some relevant compounds.
There is a significant loss of total phenol (TPC) from raw to
cooked beans of up to 66 % (Online resource 2). The amount of
phenols in our samples is similar to previous reports that
analyzed a variety of cooked black beans [12]. The total
A B
D E
F G
Fig. 1 Area of β-cells and representative histological micrographs of the
pancreas from rats treated with beans N8025 before and after induction of
diabetes. Healthy control group at first stage (a); Diabetic control group at
first stage (b); Preventive-treatment group at first stage (c); Healthy control
group at second stage (d); Diabetic control group at second stage (e);
tannins (TT) content shows a significant drop, with only
6.5 % of the compounds remaining after cooking. For total
anthocyanins (TA) content, the loss from cooking is 59 %.
Tannins and anthocyanins are reduced when exposed to heat;
they form insoluble complexes with protein that are not
extracted [17, 18].
The individual phenolic characterization with HPLC-DAD
revealed differences between the two conditions. The raw
N8025 beans had a significantly higher amount of the com-
pounds (Online resource 2) with a loss after cooking in
which almost the entire amounts of syringic and benzoic
acid are lost. Although cooking is necessary to eliminate
antinutritious compounds and some inhibitors, there is also
great loss of beneficial compounds. However, cooked beans
maintain sufficient phenols to prevent oxidative stress in
controlling diabetes [10].
Antidiabetic Effect
The STZ-induced diabetes model was used to evaluate the
beneficial effect of cooked beans. One week after the STZ
induction, there was an increase in the level of blood glucose
that was at least five times higher than that of the healthy
control (Online resource 3). The preventive-treatment group
showed the highest glucose levels at this stage. However,
one week later, it showed glucose levels similar to the other
STZ-induced groups (Online resource 3). The high blood
glucose levels of the preventive-treatment group could be
H
a
b
b
K
a
b
c
c
Preventive-treatment group at second stage (f); Treatment group at second
stage (g); Mean area of β islets at first experimental stage (h); Mean area of
β islets at second experimental stage (k). The scale bar specifies 100 μm. a,
b, c
Indicates significant statistical difference (P <0.05) between treatments
analyzed with Tukey’s test
 Plant Foods Hum Nutr

Table 1 Serum lipid profile in healthy and diabetic rats fed with beans As a source of antioxidants, beans have been used to protect
N8025 (25%)
the kidney and liver against oxidative stress in STZ-induced
TAG1 TC1 LDL1 HDL1 diabetic rats [7, 21]. It may be that the same mechanism
improves the hyperglycemic condition observed for bean
Healthy 107.6±11.7b 62.1±2.1c 39.8±3.1b 22.3±0.3b extracts, but its effect on the pancreas has not been assessed.
control
Diabetic 158.9±21.3a 120.9±1.1a 94.6±1.6a 26.2±2.2b
The study was divided in two phases: the first was
control evaluated one week after induction of diabetes, and the second
Preventive- 140.2±14.4a 63.4±1.0c 38.3±0.7b 25.1±0.9b four weeks later. Illustrative microphotographs of pancreatic
treatment islets are shown in Fig. 1. In the first phase, we observed the
Treatment 124.1±17.7ab 84.7±3.2b 44.7±3.3b 39.9±0.5a
destruction of pancreatic β-cells with the diabetic control
Values are presented as means ± SE. a, b indicates significant statistical animals (Fig. 1b), but also a degree of protection in animals
difference (P <0.05) between treatments analyzed with Tukey’s test. from the preventive group (Fig. 1c). There is no statistical
1
TAG triglycerides, TC total cholesterol, LDL low density lipoprotein significance in the cell area between the two groups, but there
cholesterol, HDL high density lipoprotein cholesterol, all values were
is a trend for higher cell volume in this group (Fig. 1h). The
expressed as mg/dL
protection is in contrast with the glucose levels determined
one week after the induction with STZ. This result suggests
attributed to the phenolic compounds in the diet that might that the conservation of the β-cell mass may not be sufficient
amplify the cytotoxic effect of STZ. Since the induction was to improve glycemic control. Also, it indicates that, in addition
carried out four weeks after feeding with beans began, and the to the damage induced by STZ [7, 20], there may be other
damage induced by streptozotocin is due to its potent oxidant mechanisms responsible for the decrease in insulin levels or in
capacity, the increased levels of phenolic compounds might its biological activity [21].
account for this observation [19, 20]. However, further The samples obtained from the second phase were
research needs to be conducted to clarify the mechanism. Four obtained 5 weeks after the induction of the diabetes (see
weeks after the induction of diabetes (week 8) the preventive- Fig. 1). There was almost a complete destruction (97%) of
treatment group showed blood glucose levels similar to the β-cell islets in the diabetic control animals (Fig. 1e and k).
diabetic control. The treatment group showed a decrease of A certain degree of protection can be seen in tissues from
around 20 % in blood glucose level at the end of the experi- the treatment group (37%) and a trend of conservation of ß-
ment, which is statistically different from the diabetic control cells for the preventive group (12%) (Fig. 1f, g and k). The
(Online resource 3). This suggests that once the diabetes fully treatment group shows a higher preservation of islet area at
develops, the supplementation of the diet with bean may help the end of the experiment, and most importantly, it seems to
to reduce the hyperglycemic state. The beneficial effect of lead to a significant decrease in glucose levels (Online
beans in the diet is not seen in the preventive-treatment group, resource 3). These results suggest that the inclusion of beans
even though this group shows a trend to lower glucose levels in the diet can protect against the destruction of the ß-cells,
than observed with the diabetic control group, perhaps due to thus improving the glycemic state. The low protection found
the initial high glucose levels. for animals from the preventive study suggests that the
active compounds may have opposite effects. Certain poly-
Histological Analysis of the Pancreas phenolic extracts have demonstrated a similar effect with
STZ-induced diabetic rats [23] that might be due to the
The effect of antioxidant compounds against pancreatic chronic exposure to the bean diet that may cause long-
destruction was studied as previously described [21, 22]. term damage rather that help in counteracting the disease.

Table 2 Serum inflammatory markers TNF-α and IL-1β for healthy and diabetic rats fed with beans N8025 (25%) before and after induction of diabetes

TNF-α1 IL-1β1

1st stage 2nd stage 1st stage 2nd stage

Healthy control 10.9±0.6Ac 9.5±1.8Ac 120.0±10.0Ab 110.0±6.6Ac

Diabetic control 75.4±0.7Aa 85.1±6.1Aa 300.0±26.5Ba 790.0±90.7Aa
Preventive-treatment 58.1±1.5Bb 74.0±2.5Aa 115.0±7.7Bb 386.0±60.7Ab
Treatment NQ 40.5±1.9b NQ 400.0±60.0b

Values are presented as means ± SE. a, b Indicates significant statistical difference (P <0.05) between treatments analyzed with Tukey’s test. A, B Indicates
significant statistical difference (P <0.05) among each cytokine analyzed with Tukey’s test. 1 Values were expressed as pg/ml. NQ not quantified
Plant Foods Hum Nutr
Lipid Profile
After an increase in hyperglycemia, other metabolic changes
occur, one of which is an increase of lipids concentration.
The lipid profile was measured for all groups (Table 1), and
as expected, TAG showed an increase of around 50% in the
diabetic control group, compared to that for healthy animals.
The two groups fed with bean diets did not reach the same
TAG levels. Lower TAG levels were observed for the treat-
ment group with a decrease of 22%, while the preventive
group had a decrease of around 12% (Table 1). The levels of
circulating TAG are consistent with the glucose levels of all
four groups.
The induction of diabetes increases cholesterol levels
(Table 1). Of the two diabetic groups that were fed the bean
diet, the treatment group has a lower TC level by 30%,
while the preventive group has a decrease of 47%. It seems
that N8025 bean flour has the potential to reduce total
cholesterol levels. In agreement with this change, LDL
levels changed proportionally. The main value of LDL was
obtained for the diabetic control group with an increase of
more than a factor of two, related to that of the healthy
control group (Table 1). The values for the other two dia-
betic groups that were fed bean were statistically similar to
the healthy control. No difference was observed between
healthy, diabetic, and preventive-treatment groups for the
HDL levels. However, HDL, a beneficial cardiovascular
marker, increased in the treatment group (Table 1).
According to previous reports, sustained hyperglyce-
mia causes structural changes in LDL. As a consequence,
LDL particles are not recognized by their cellular recep-
tor, and thus remain longer in the blood stream [24].
N8025 bean consumption exerts a positive effect by
decreasing the LDL level for both the preventive and
the treatment groups. Data from another study suggest
that apo B/E receptor activity is induced in the liver of
bean-fed rats, as well as in peripheral tissues, presumably
as a consequence of the marked stimulation of biliary
cholesterol output and of the significantly higher rate of
bile salt synthesis induced by a bean diet [25].
Inflammatory Markers
If the cells are subjected to a continuous hyperglycemic stage,
there is an increase in the oxidative stress that is responsible for
the damage induced by diabetes. In addition to this, there is a
rise in the inflammatory condition. Pancreatic destruction has
been related to inflammation, due to an increase in the produc-
tion of pro-inflammatory molecules such as the tumor necrosis
factor-α (TNF-α) and interleukine-1β (IL-1β) [22]. We mea-
sured these two cytokines to complete our understanding of the
development of diabetes and the effect of the supplementation
of diet with cooked beans.
The measure was carried out at the end of the two stages.
The levels of the two cytokines increased considerably in the
diabetic control at the end of the first stage (Table 2): TNF-α
increased by nearly 7-fold, and IL-1β by about 2.5-fold,
compared to the control. The preventive group exhibits values
of TNF-α that are lower than the diabetic control and higher
than the healthy, both statistically different, and not statistical-
ly different for IL-1ß.
For the second stage, the diabetic group has the highest
values of both cytokines: the levels are 9- and 7- fold higher for
TNF-α and IL-1ß, respectively. From the first to the second
stage, the increases in the groups fall between reasonable
values, almost stable for the healthy control and with a signif-
icant increase for the diabetic control. The treatment groups
have values that are lower than the diabetic control (Table 2).
It has been reported that Phaseolus angularis can decrease
the level of inflammatory products such as prostaglandins
(PGE2) and nitric oxide. The effect could be through the inhi-
bition of the nuclear factor κB (NFκB), nitric oxide inducible
synthase (iNOS), and cyclooxygenase-2 (COX-2) [26]. Previ-
ous studies reported that acetone extracts from black bean
(Phaseolus vulgaris L.) inhibit inflammation mediators such
as COX-1 and COX-2, which is mainly attributed to antioxi-
dants [27]. Also, an ethanol extract was able to decrease TNF-α
and increase the anti-inflammatory cytokine adiponectin [28].
Conclusion
These data provide biological evidence to support the sug-
gestion to increase the ingestion of beans. Although cooking
causes an important loss of nutraceutical components, those
that remain are sufficient to help maintain ß-cell mass during
hyperglycemia and to improve the glycemic status and its
utility in controlling diabetes.
Conflict of interest The authors declare that they have no conflict of
interest.
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