Hindawi Publishing Corporation

Oxidative Medicine and Cellular Longevity

Volume 2016, Article ID 1398298, 12 pages
http://dx.doi.org/10.1155/2016/1398298

Research Article
Phenolic Composition and Antioxidant and Antiproliferative
Activities of the Extracts of Twelve Common Bean
(Phaseolus vulgaris L.) Endemic Ecotypes of Southern Italy
before and after Cooking

Maria Neve Ombra,1 Antonio d’Acierno,1 Filomena Nazzaro,1

Riccardo Riccardi,2 Patrizia Spigno,2 Massimo Zaccardelli,3 Catello Pane,3
Mena Maione,1 and Florinda Fratianni1
1
Institute of Food Science, CNR-ISA, Via Roma 64, 83100, Avellino, Italy
2
Azienda “F. Marsocci”, Via Varignano 7, 80011 Acerra, Italy
3
CREA, Via dei Cavalleggeri 25, 84098 Pontecagnano, Italy

Correspondence should be addressed to Filomena Nazzaro; mena@isa.cnr.it

Received 23 September 2016; Accepted 25 October 2016

Academic Editor: Ilaria Peluso

Copyright © 2016 Maria Neve Ombra et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Beans are important dietary components with versatile health benefits. We analysed the extracts of twelve ecotypes of Phaseolus
vulgaris in order to determine their phenolic profiles, antioxidant activity, and the in vitro antiproliferative activity. Ultra-
performance liquid chromatography with diode array detector (UPLC-DAD) admitted us to detect and quantify some known
polyphenols, such as gallic acid, chlorogenic acid, epicatechin, myricetin, formononetin, caffeic acid, and kaempferol. The
antioxidant activity (AA) ranged from 1.568 ± 0.041 to 66.572 ± 3.197 mg necessary to inhibit the activity of the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical by 50% (EC50 ). The extracts, except those obtained from the nonpigmented samples, were capable
of inhibiting the proliferation of the human epithelial colorectal adenocarcinoma (Caco-2) cells, human breast cancer cells MCF-7,
and A549 NSCLC cell line. Cultivars differed in composition and concentration of polyphenols including anthocyanins; cooking
affected the antioxidant activity only marginally. Qualitative and quantitative differences in phenolic composition between the
groups of beans influenced the biological activities; on the other hand, we did not find significant differences on the biological
activities within the same variety, before and after cooking.

1. Introduction delphinidin and cyanidin, that overall attribute them a very

strong antioxidant and antiradical activities [3]. Polyphenols
Among legumes, common beans (Phaseolus vulgaris L.) are essentially present in the seed coats and in minor amount
are widely consumed throughout the world. They play a in cotyledons.
significant role in human nutrition, being an important The beneficial effects of polyphenols on human health are
source of plant proteins, minerals, and certain vitamins and expressed primarily through the reduction of the oxidative
exhibit, for this reason, high nutritional value. In last years, stress [4]. Some polyphenols are also able to exert antiapop-
bioactive effects, associated with the fibres, polyphenols, totic, antiaging, as well as anticarcinogenic activity, overall
and other beans components related to human health have inhibiting the cell proliferation processes [5, 6].
gained attention [1, 2]. Beans contain substantial amount of Epidemiological studies have shown a strong link between
phenolic acids and flavonoids; some cultivars (red, black, and the consumption of legumes and prevention of cancer risk,
blue-violet coloured beans) show also anthocyanins, such as as well as the reduction of diabetes and cardiovascular risk
2 Oxidative Medicine and Cellular Longevity

through a normalization of the blood lipid and glucose colorimetric method, as described by Singleton and Rossi
profile [7]. Wedick et al. (2012) demonstrated that, among Jr. [11]. Briefly, 50 𝜇L of extract was added to 50 𝜇L of
flavonoids, higher intakes of anthocyanins could be signifi- Folin-Ciocalteu reagent in 800 𝜇L of distilled water. The
cantly associated with a lower risk of type 2 diabetes (risk: reaction was neutralized with a sodium carbonate solution
0.85) [8]. (20 g/100 mL). After incubation for 2 h at room temperature,
Legumes are generally eaten without the elimination of the absorbance at 𝜆 = 760 nm was determined using a Cary
the coat and in any case after cooking. Oroian and Escriche UV/Vis spectrophotometer (Varian, Palo Alto, CA, USA).
(2015) reported the antioxidant effect and total phenolic Quantification was based on a standard curve generated using
content of common beans, showing that pigmented beans had gallic acid. The results were expressed as 𝜇g of gallic acid
generally major antioxidant effect and higher amount of total equivalents (GAE)/g of DW samples ± standard deviation
polyphenols, with respect to the nonpigmented ones [9]. Due (SD).
to their particular geographical characteristics, the territory
of Southern Italy shows many different environments. During 2.3. Total Flavonoids. Total flavonoids (TF) were spectropho-
the past centuries, local farmers selected various bean seeds tometrically determined at 510 nm following the method of
after their discovery in South America and diffusion in Zhishen et al. [12] with some modifications. The extract
Europe. A so long activity of selection produced a small (50 𝜇L) was added to distilled water followed by 5% NaNO2
number of selected local cultivars, with unique characteris- and after 5 min by AlCl3 10%. After further 5 min, the reaction
tics. Studies regarding the effect of thermal processing on the mixture was treated with 0.2 mL of 1 mM NaOH. Finally,
health relevant functionality of beans are limited, especially the reaction mixture was diluted to 1 mL with deionised
with respect to the traditional cultivars present in those areas water and the absorbance was measured at 𝜆 = 510 nm
where the model of the Mediterranean diet is still strong. An (Varian, Palo Alto, CA, USA). TF amounts were expressed
improved information of the chemical composition and bio- as 𝜇g quercetin equivalents/gram of DW samples ± standard
logical properties of local bean cultivars could help to define deviation (SD).
and valorise those with a greater nutraceutical potential to
suggest to consumers. 2.4. Anthocyanin Content. The amount of anthocyanins was
Starting from such assumptions, aim of this study was to determined by the differential pH method [13]. Absorbance
study the polyphenols (including total flavonoids and antho- was measured in a Cary UV/Vis spectrophotometer (Varian,
cyanins), the antioxidant activity, and the antiproliferative USA), simultaneously at 𝜆 = 510 nm and 𝜆 = 700 nm
activity of the extracts obtained from some nonpigmented, in buffers of pH 1.0 and 4.5, using the formula 𝐴 =
red, speckled, and dark beans endemic ecotypes of Southern (𝐴510 − 𝐴700)pH1.0 − (𝐴510 − 𝐴700)pH4.5 . A molar absorp-
tion of 26,900 mol/cm was used for cyanidin-3-glucoside
Italy. The study was carried out before and after the cooking
(molecular weight of 449.2 g/mol). Results were expressed
domestic process.
as micrograms of cyanidin-3-glucoside equivalents/g of DW
samples ± standard deviation (SD).
2. Materials and Methods
2.5. DPPH Radical-Scavenging Activity. The free radical-
Twelve varieties of common bean (Phaseolus vulgaris L.) scavenging activity was determined using the stable radical
were collected in various rural communities from Campania 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) [14]. The analy-
and Basilicata regions of Southern Italy (Figure 1). Samples sis was performed in microplates by adding 15 𝜇L of extract
were divided into four bean groups based on the colour: to 300 𝜇L of a methanolic DPPH solution (6 × 10−5 M).
nonpigmented beans, red beans, speckled beans, and dark Next, the absorbance at 𝜆 = 517 nm was spectropho-
beans; each group included three cultivars (Table 1). Dried tometrically measured (Cary 50 MPR, Varian, USA). The
beans were stored in the dark prior to extraction. absorbance of DPPH without antioxidant (control sample)
was used for baseline measurements. The scavenging activity
2.1. Sample Preparation. Samples (10 g) were rehydrated (1 : 5, was expressed as the 50% effective concentration (EC50),
w/v) for 24 hours; the water was discharged and 4 volumes which was defined as the sample amount (mg) necessary
of acetone were added. After 2 h of incubation at 4∘ C and to inhibit DPPH radical activity by 50% during 60 min of
centrifugation at 11,600 ×g (Biofuge, Beckman, Cassina de incubation. These experiments were performed in triplicate,
Pecchi, Italy), the supernatants were recovered and stored at and the results were expressed as the mean values ± standard
4∘ C. Pellets were treated again with one volume of acetone deviation.
and incubated for 1 h at 4∘ C; the two supernatants were
pooled, filtered, and, after a complete evaporation, stored at 2.6. Phenol Antioxidant Index (PAOXI). As suggested by
−20∘ C in the dark, until the analyses were performed [10]. The Vinson et al. [15] the phenol antioxidant index (PAOXI) can
cooking of beans was performed with deionised water (1 : 5, be used to estimate a combined measure of the quality and
w/v), in a home pressure cooker, for 20 min after the constant quantity of antioxidants present in a sample. In this study,
output of steam by the pressure safety valve. Cooked beans it was calculated by dividing the DPPH radical-scavenging
were immediately cooled, and the extracts were prepared as activity of the sample (𝜇mol of DPPH inhibited/mg) for the
previously described. total phenol concentration (mg/g) [16].

2.2. Total Polyphenol Content. Total polyphenols (TP) of 2.7. Chromatographic Analysis. An ACQUITY Ultra-
the extracts were determined using the Folin-Ciocalteu Performance LC system (Waters) linked to a PDA 2996
Oxidative Medicine and Cellular Longevity 3

Table 1: Sample classification.

Raw sample Cooked sample

(1a) Not pigmented beans: Bianco Acerra (BA) (1a󸀠 ) Not pigmented beans: Bianco Acerra (BAc)
(1b) Not pigmented beans: Dente de Morto (DM) (1b󸀠 ) Not pigmented beans: Dente de Morto (DMc)
(1c) Not pigmented beans: Volturara (V) (1c󸀠 ) Not pigmented beans: Volturara (Vc)
(2a) Red beans: Cannellino Rosso (CR) (2a󸀠 ) Red beans: Cannellino Rosso (CRc)
(2b) Red beans: O Russ (OR) (2b󸀠 ) Red beans: O Russ (ORc)
(2c) Red beans: Zampognaro (Z) (2c󸀠 ) Red beans: Zampognaro (Zc)
(3a) Speckled beans: Pettilanculo (P) (3a󸀠 ) Speckled beans: Pettilanculo (Pc)
(3b) Speckled beans: Sanghellatto (S) (3b󸀠 ) Speckled beans: Sanghellatto(Sc)
(3c) Speckled beans: Screziato impalato (Si) (3c󸀠 ) Speckled beans: Screziato impalato (Sic)
(4a) Dark beans: Nero Acerra (NA) (4a󸀠 ) Dark beans: Nero Acerra (NAc)
(4b) Dark beans: Nero Frigento (NF) (4b󸀠 ) Dark beans: Nero Frigento (NFc)
(4c) Dark beans: Nero Caposele (NC) (4c󸀠 ) Dark beans: Nero Caposele (NCc)
Cooked samples are indicated with “c.”

BA-DM-V

CR-OR-Z

P-S-SI

NA-NF-NC

Figure 1: Photo of 12 varieties of beans (Phaseolus vulgaris L.) analyzed. Samples were classified into four groups: nonpigmented beans, red
beans, speckled beans, and dark beans. Nonpigmented beans: Bianco Acerra (BA), Dente di morto (DM), and Volturara (V). Red beans:
Cannellino Rosso (CR), ‘O Russ (OR), and Zampognaro (Z). Speckled beans: Pettilanculo (P), Sanghellatto (S), and Screziato impalato (Si).
Dark beans: Nero di Acerra (NA), Nero di Frigento (NF), and Nero di Caposele (NC).

photodiode array detector (Waters) was used for ultra- following the method of Fratianni et al. [17]. The mobile
performance liquid chromatography analyses. The Empower phase consisted of solvent A (7.5 mM acetic acid) and solvent
software controlled the instruments and acquired and B (acetonitrile) at a flow rate of 250 𝜇L min−1 . Gradient
processed the data. The extracts and standards (previously elution was employed, starting with 5% B for 0.8 min; then
dissolved in methanol) were filtered (0.45 𝜇m; Waters) before 5–20% B over 5.2 min; isocratic 20% B for 0.5 min; 20–30%
analysis. The analyses were carried at 30∘ C using a reversed B for 1 min; isocratic 30% B for 0.2 min; 30–50% B over
phase column (BEH C18 , 1.7 𝜇m, 2.1 100 mm; Waters) 2.3 min; 50–100% B over 1 min; isocratic 100% B for 1 min;
4 Oxidative Medicine and Cellular Longevity

and 100–5% B over 0.5 min. At the end of this process, the significance was accepted at 𝑝 < 0.05. The PC software
system equilibrated the column under the initial conditions “Excel Statistics” was used for the calculations. Principal
for 2.5 min. Component Analysis (PCA) was used to relate values of
biological activities to phenolic composition, using the free
2.8. Determination of In Vitro Cancer Cell Viability software environment for statistical computing and graphics
R (https://www.r-project.org/). For cell assays, the EC50
2.8.1. Cell Culture and Extracts. MCF-7, A549, and Caco-2 values were calculated using ED50plus v1.0 online software.
cell lines were obtained from the American Type Culture
Collection (ATCC, Rockville, MD). Cells were grown in 3. Results and Discussion
Dulbecco’s modified eagle’s medium (DMEM) supplemented
with 10% of heat-inactivated foetal bovine serum (FBS), peni- 3.1. Polyphenol Content. The content of total polyphenols
cillin (0.010 U L−1 ), streptomycin (10 mg L−1 ), and sodium (TP) present in the extracts of beans is shown in Table 2.
pyruvate 0.0002 M. Cultures were incubated in the presence Results, expressed as 𝜇g GAE/gr (dw of seeds), showed
of 5% CO2 at 37∘ C and 100% relative humidified atmosphere. significant differences (𝑝 ≤ 0.01) among samples; on the
Several concentrations of extract were dissolved in DMEM other hand, such differences were not significant (𝑝 >
medium. The culture media were evaluated in terms of pH 0.05) within the same group of beans. Maximal quantitative
and precipitation of components, in order to avoid possible differences in TP were obtained between the extracts of
cytotoxic effects due to changes in these factors on the cellular white beans (ranging from 135.04 to 275.4) and speckled
microenvironment. Only the concentrations that did not beans (ranging from 1061.9 to 1249.7), with an even ratio of
change the culture media conditions were selected for further about 1 : 10. In the extracts of red and black beans, values of
assays. TP were intermediate, in agreement to the results reported
by Heimler et al. [19], which studied other Italian varieties
2.8.2. Crystal Violet Assay. The cells were plated (10 × of Phaseolus vulgaris L. An exception was represented by
103 /well) in 48-well plates in complete medium. After 24 h, the extract of Cannellino Rosso (CR) (TP = 229.70) which,
the medium was removed and replaced on the first day although belonging to the red group, exhibited an amount
with the same medium (control) or supplemented with of polyphenols comparable to that found in nonpigmented
various extracts doses. Cells were observed with an inverted beans. The colour of this variety was more clear compared to
microscope every 24 h, to identify morphological changes, the other red beans (Figure 1).
toxicity, or cell death. The percentage of cell proliferation was The extracts obtained from the cooked beans showed
estimated on the fourth day by a colorimetric assay reported a lower amount of TP with respect to the corresponding
by Kueng et al. [18] modified as follows: cells were fixed raw samples, except those obtained from the nonpigmented
for 20 min at room temperature with 1% paraformaldehyde beans, for which the amount of polyphenols remained similar
(PFA), stained with 0.1% crystal violet in 20% methanol for after cooking. For example, taking into consideration the
20 min, washed with PBS, solubilized with 10% acetic acid, cultivar Bianco di Acerra, the amount of TP remained
and read at 595 nm in a microplate reader (Cary 50 MPR, virtually the same, before and after the thermal treatment
Varian, USA). The experimental conditions were tested in (275.442 and 241.782, resp.).
triplicate, and separate experiments were performed on at
least three separate occasions. 3.2. Determination of Flavonoids. Usually, flavonoids and
other phenolic compounds are typically stored in the seed
2.8.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium coat due to their antipathogen and antifeeding activities;
Bromide (MTT) Reduction Assay. Cells were seeded in 96- furthermore, such localization assures the best protection of
the seed from external attacks (pathogens, insects, etc.) [20].
well microplates at a density of 5 × 103 cells/well and grown for
Many of the flavonoids that give rise to the coat colour of
24 h at 37∘ C in 5% CO2 prior to the addition of test samples. beans may also provide positive health benefits as antioxi-
The cells were treated with different sample concentrations dants. This class of polyphenols was present in all samples
dissolved in Dulbecco’s phosphate buffered saline (PBS). analysed, being particularly abundant in the coloured beans,
After 48 h of incubation, cell viability was determined using with a very similar trend to that of the polyphenols, as
a colorimetric MTT assay. Cell survival (%) was measured shown in Table 2. The extracts of white beans showed a
as the reduction of MTT in formazan at 550 nm. Triton X- low content of flavonoids, not more than 58.73 𝜇g/QE. The
100 (10 𝜇L of 10% solution) was used as a positive control. highest quantities of flavonoids were found in the extracts of
Untreated cells (vehicle alone) were chosen as the negative speckled beans as well as in that of the red cultivar ‘O Russu
control. The controls and samples were assayed in tripli- (OR). The extracts obtained from the three speckled beans
cate for each concentration and replicated three times. The Pettilanculo (P), Sanghellatto (S), and Screziato impalato (SI)
absorbance values were converted into percentages of cell showed 925.654 𝜇g/QE, 910.551 𝜇g/QE, and 703.336 𝜇g/QE,
viability using the following formula: Cell viability = Abs respectively; the extract of cultivar Nero di Acerra (NA) was
sample/Abs control × 100. the richest in terms of content in flavonoids among dark
beans analysed (542.337 𝜇g/QE).
2.9. Statistical Analysis. Data were expressed as mean ±
standard deviation of triplicate measurements. Analysis 3.3. Determination of Total Anthocyanins. As expected, a
of variance (ANOVA) was used to compare results and higher content of anthocyanins was observed in the extracts
Oxidative Medicine and Cellular Longevity 5

Table 2: Total polyphenols (PT), antioxidant activity, flavonoids (FT), and anthocyanin contents in the bean extracts. The concentration of
total polyphenols was expressed as 𝜇g equivalents of gallic acid/g of DW sample; antioxidant activity was expressed as EC50 (mg/mL DPPH);
flavonoids were expressed as 𝜇g equivalents of quercetin/g of DW sample; the content of anthocyanin content was expressed as 𝜇g equivalents
of cyanidin-3-glucoside/g of DW sample. Data are the mean values of three independent experiments (±standard deviation).

Polyphenol Antioxidant Flavonoids Anthocyanin

content activity content content
(𝜇g GAE/gr (DW)) (mg × mL di DPPH) (𝜇g (QE)/gr (DW)) (𝜇g (C3GE)/gr (DW))
(BA) 275.442 ± 58.621 23.619 ± 0.532 54.733 ± 3.316 0
(DM) 135.037 ± 6.316 55.217 ± 5.178 58.731 ± 2.471 0
(V) 165.524 ± 12.77 28.826 ± 2.329 51.511 ± 1.422 0
(BA-c) 241.782 ± 54.213 61.605 ± 8.087 50.992 ± 4.804 0
(DM-c) 211.907 ± 37.017 50.133 ± 3.786 34.323 ± 4.798 0
(V-c) 126.29 ± 20.212 66.572 ± 3.197 49.483 ± 5.883 0
(CR) 229.698 ± 3.909 10.619 ± 0.211 100.151 ± 9.367 3.015 ± 0.691
(OR) 1205.446 ± 23.884 1.722 ± 0.013 878.749 ± 58.878 8.864 ± 0.773
(Z) 997.774 ± 90.482 1.927 ± 0.036 562.378 ± 48.128 10.706 ± 0.561
(CR-c) 182.275 ± 23.009 27.291 ± 3.351 116.189 ± 2.822 1.148 ± 0.068
(OR-c) 621.338 ± 27.434 6.022 ± 0.145 360.417 ± 4.864 5.323 ± 0.248
(Z-c) 687.874 ± 25.338 4.849 ± 0.045 202.396 ± 11.1 6.113 ± 0.763
(P) 1249.71 ± 29.941 1.583 ± 0.047 925.654 ± 90.209 6.314 ± 1.285
(S) 1061.935 ± 13.603 1.568 ± 0.041 910.551 ± 137.472 3.629 ± 0.193
(Si) 1150.474 ± 15.993 1.813 ± 0.056 703.336 ± 121.5 7.255 ± 1.794
(P-c) 607.606 ± 8.486 5.052 ± 0.171 332.618 ± 6.633 2.388 ± 0.796
(S-c) 565.326 ± 58.081 3.82 ± 0.003 192.021 ± 21.969 1.338 ± 0.372
(Si-c) 622.14 ± 74.146 6.253 ± 0.116 245.306 ± 22.634 3.963 ± 0.456
(NA) 604.23 ± 53.995 5.161 ± 0.352 244.698 ± 14.524 29.702 ± 3.327
(NF) 601.269 ± 52.156 4.346 ± 0.088 293.928 ± 9.119 18.909 ± 2.457
(NC) 1291.62 ± 40.16 4.347 ± 0.609 542.337 ± 16.454 63.278 ± 1.547
(NA-c) 359.844 ± 28.37 7.996 ± 0.042 112.927 ± 2.86 5.63 ± 0.799
(NF-c) 240.553 ± 23.097 14.528 ± 0.803 99.694 ± 4.163 6.939 ± 0.608
(NC-c) 436.626 ± 50.194 6.78 ± 0.041 193.103 ± 9.974 10.508 ± 1.206

of dark beans, while lower amounts were found in those extracts were sufficient to inhibit at 50% of the activity of
of speckled and red samples, except Zampognaro (Z) bean 1 mL of the free radical DPPH (Table 2). Speckled beans
(10.706 𝜇g C3GE/gram) (Table 2). The extract of the dark showed the highest antioxidant activity (EC50 not exceeding
bean Nero di Caposele (NC) showed the highest value 1.813 mg/mL), followed by red beans (except for CR) and
(63.278 𝜇g C3GE/gram) of anthocyanins; moreover, this cul- black beans (EC50 not superior than 5.2 mg/mL). As expected,
tivar proved to be also the richest with regard to total polyphe- the extracts of nonpigmented varieties exhibited the low-
nols and flavonoids among the dark cultivars analysed. It est antioxidant activity (with EC50 values ranging between
should be emphasized that the anthocyanins in the beans are 23.62 mg/mL and 55.21 mg/mL). Cooked beans, in spite of
exclusively present in the peel [21]. Therefore it is sufficient a loss of total polyphenols, disclosed intriguing results. To
that the seeds are smaller to obtain a higher anthocyanin estimate the combined measure of the quality and quantity of
value, as we observed in our test as regard as cultivar NC. antioxidants present in the sample, we adopted the so-called
Cooking lowered the content of anthocyanins present in the phenol antioxidant index (PAOXI) as suggested by Vinson et
samples; however, some cultivars (Z among speckled and NC al. [15]. In our study, such index was calculated by dividing the
among dark cultivars) maintained a content of anthocyanin DPPH radical-scavenging activity of the sample (expressed
of 6.11 𝜇g C3GE/gram and 10.508 𝜇g C3GE/gram, respec- in this case as 𝜇mol of DPPH inhibited/gram of product)
tively. with respect to the total polyphenol concentration (mg/g).
This was possible by converting the values obtained in the
3.4. Antioxidant Activity. The antioxidant effects in bean antioxidant activity in mg, to 𝜇M DPPH/mg sample. The
extracts were investigated by DPPH test. Overall, the samples highest PAOXI value (36.25) was exhibited by the extract of
exhibited relevant antioxidant qualities and few mg of the the speckled bean Sanghellatto (S); therefore, the extract of
6 Oxidative Medicine and Cellular Longevity

Table 3: The phenol antioxidant index (PAOXI) (𝜇mol of DPPH apparatus, on the basis of known standard. The data are
inhibited/mg) by the total phenol concentration (mg/g). Data are separated according to the groups.
the means values of three independent experiments (± standard The profiles of the extracts of nonpigmented beans were
deviation).
very simple; in fact, we found only few peaks, present in the
PAOXI initial part of the chromatogram. The extract of the variety
Raw sample Cooked sample Dente di Morto DM showed even a minor number of peaks
(1a) (BA) 9.486 4.016 with respect to the other two nonpigmented beans BA and
Q analysed (chromatogram not shown). Despite a few peaks
(1b) (DM) 8.081 5.687
viewable, the sensitivity of the equipment still allowed us to
(1c) (V) 12.987 7.215
detect and identify several known polyphenols, even if they
(2a) (CR) 24.61 12.076 were present in small quantities in the nonpigmented beans
(2b) (OR) 28.71 16.05 (Table 4(a)). Polyphenol profiles of the extracts of speckled,
(2c) (Z) 31.344 18.045 red, and dark beans were richer and more complete. Some
(3a) (P) 30.187 19.574 polyphenols were present only in the extracts giving rise from
(3b) (S) 36.25 27.8 the pigmented beans. For example, myricetin was identified
(3c) (Si) 29.002 15.434
only in the extracts of the pigmented beans: it was present
in the extract of raw bean NC (1.986 𝜇g/gram, Table 4(d))
(4a) (NA) 19.252 20.918
and in the extracts of Z and Si after cooking (3.35 𝜇g/gram
(4b) (NF) 23.003 17.206 and 0.218 𝜇g/gram, Tables 4(c) and 4(b), resp.). Overall, our
(4c) (NC) 10.709 20.297 results were in agreement with dosage of polyphenols previ-
ously reported for beans, especially as regards flavonoids [22,
23]. We analysed more extensively those flavonoids already
the dark cultivar Nero di Caposele exhibited the lowest PAOXI reported in literature for their antiproliferative activity [24],
value (10.79) among coloured beans (Table 3). This simple and we found appreciable amount of some flavones and/or
calculation could help in clarifying about the relationship flavonoids, such as formononetin, genistein, quercetin 3,4-
between polyphenols and antioxidant activity that is not diglucoside, spiraeside, and hyperoside, in the extracts of red
always so direct. samples (Table 4(b)). The extract of Cannellino Rosso, while
Although it was possible to establish a clear correlation containing a modest amount of total polyphenol with respect
between the levels of polyphenols and the free radical- to the extracts of the other two red cultivars, exhibited a
scavenging properties of the extracts, we observed some polyphenol profile absolutely well correlated with respect to
diversity, which could be ascribable to the differences the extracts of the other two red varieties. It should also be
between their components constituting total polyphenols. It highlighted that the decrease of total polyphenols after the
is known that there are a variable number of combinations cooking of beans did not affect the amount of flavonoids,
of the compounds for each sample and that the antioxi- such as genistein and formononetin. Formononetin is a
dant potential of these individual components is variable. typical bioactive isoflavone of red clover plant with potent
From the analysis of total polyphenols, total flavonoids, and pharmacological activities, including antioxidant, antiviral,
antioxidant activity, we observed that the extracts of the dark antitumor, and cardio protective effects [25]. In addition,
bean Nero di Caposele (NC) showed the highest amount this isoflavone is capable of inhibiting the proliferation,
of total polyphenols; however, speckled Screziato impalato inducing apoptosis in human cancer cells, such as breast
bean (SI), although containing slightly less polyphenols, cancer, prostate tumour, and osteosarcoma [26, 27]. A study
exhibited higher flavonoid content, and its PAOXI index by HPLC performed on 62 wild and weedy Mexican bean
was significantly higher (10.79 versus 29.0). This reinforced ecotypes identified kaempferol as one of the main flavonoids,
the idea that the antioxidant potential could be strongly assuming that its quantitative variation could be more related
linked to the content of flavonoids. Cooking affected the to the genotype than to the seed coat colour [28]. In our
antioxidant activity; for some varieties (e.g., NA and NF) the experiments, flavonols, such as quercetin and kaempferol,
PAOXI values were similar to those obtained before cooking; were found both in raw and in processed beans. Likewise, we
indeed in some cases (NC) a higher PAOXI index was found kaempferol in the extracts of red cultivar Zampognaro
observed, suggesting that the thermal processing reinforced (Z) and in those of the two speckled P and Si cultivars (Tables
their antioxidant efficacy. 4(b) and 4(c)). Kaempferol is one of the phenolic compounds
more studied for its antimutagenic and anticarcinogenic
3.5. Identification of Polyphenols. The UPLC analysis pro- activity both in vitro and in vivo [29]; its concentration in
vided an overview of the main phenolic compounds in every P. vulgaris is very variable, ranging from traces < 0.2 mg/kg
sample. In Figure 2 we reported the chromatograms obtained (in Tuscany landraces) [30] to 209.4 mg/kg (in Mexican
from the extracts of four cultivars of beans, each of which cultivated varieties) [3]. Our data were more in agreement
representing a group (white, speckled, red, and dark). For with the Tuscany varieties [31]. It is to underline the presence
each one, we reported the profiles obtained before and after of these compounds in cooked beans, so that they can really
the process of cooking. Table 4 shows, organised as tabular carry out their nutraceutical properties, above all because the
form, the amount of each polyphenol recognised from the flavonols are not much affected by heat treatment [32].
Oxidative Medicine and Cellular Longevity 7

Quarantino di Volturara (Q) raw sample Quarantino di Volturara (Qc) cooked sample
1.80 1.20

1.223
1.662
1.234
1.60 1.00

0.924
1.40
1.20 0.80
1.00
AU

AU
0.60
0.933
1.711
0.80

2.854

2.854
0.60 0.40

2.103
2.108
0.40

9.684
2.488
0.20

9.683
4.049
2.383

11.902
5.451

2.428
3.360

4.986

11.907
4.040

5.448
3.356
9.988
0.20

9.978
0.00 0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
(Minutes) (Minutes)

Cannellino Rosso (CR) raw sample Cannellino Rosso (CRc) cooked sample

1.445
5.968

2.00 2.00
0.924

1.50 1.50
2.829

AU
1.254
AU

1.00 1.00

5.991
1.135
2.544

−5.378

9.684
2.054

9.529
0.50

2.834
1.744

0.50
−11.584

−5.387

−11.623
3.496

−6.469
4.106

5.785
5.019
3.688

4.663

6.712

11.836
10.032
2.199

11.263

3.355
3.682

4.918
2.648

4.654

8.627
3.951
7.269

8.620

0.00 0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
(Minutes) (Minutes)

Sanghellatto (S) raw sample Sanghellatto (Sc) cooked sample

3.00
1.073

2.50
1.070
2.50
2.00 2.00
0.935

1.50 1.50
AU
AU

2.844
2.458
2.819

2.472

1.00 1.00
1.728
3.307

2.101
9.837
5.434
4.025
3.668

6.020

3.100
3.357
4.971

0.50 0.50

9.828
4.530
4.809

5.451
4.042
1.752

11.900

11.888
11.594
11.588
6.672

3.685

5.083
2.100

7.316

4.554

6.026
6.532
5.856

8.759
8.769

0.00 0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
(Minutes) (Minutes)

Nero Frigento (NF) raw sample Nero Frigento (NFc) cooked sample
2.50
2.834

1.40
1.391
1.611
0.937

1.20 2.00
1.00 1.50
0.80
2.554

AU

1.132
AU

9.730

0.60 1.00
1.245

9.602
3.332

0.40
5.413
2.040

0.937

2.831

0.50
4.624
3.971

5.167
5.078

10.586

11.635
11.305
5.392

10.218
11.854

3.356
5.994
4.401

3.963
11.274

4.931
3.725

4.639
6.455

6.007
4.346

0.20
0.00 0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
(Minutes) (Minutes)

Figure 2: UPLC profiles of the polyphenols present into four bean groups: nonpigmented beans Quarantino di Volturara raw and cooked
(Q-Qc); red beans Cannellino Rosso raw and cooked (CR-CRc); speckled beans Sanghellatto raw and cooked (S-Sc); and dark beans Nero
Frigento raw and cooked (NF- NFc).

The extracts of the three speckled beans, in addition of formononetin; it is also to underline the presence of
to flavonoids, exhibited many caffeic acid derivatives and spiraeoside in the extract of Sanghellatto after cooking (Sc,
an appreciable amount of catechin derivatives, including Table 4(c)).
epigallocatechin (Table 4(c)). The extracts of dark beans gave a well-varied profile,
Flavonoids showed a great variability; in fact, besides the with appreciable values for formononetin and the presence
formononetin and derivatives of quercetin, they contained of quercetin glucosides 3-4 (Table 4(d)).
also genistein and daidzein, at low amounts. Antiprolifer-
ative activity of flavonoids is well known and analysed in 3.6. Anti-Proliferative Activity. The antiproliferative activity
many studies [33, 34]. After cooking, the extracts of both of the extracts was evaluated on MCF-7 breast cancer cell
Pettilanculo (P) and Sanghellatto (S) showed very high values line, A549 NSCLC cell line, and Caco-2 colon carcinoma
8 Oxidative Medicine and Cellular Longevity

Table 4: Chromatographic profile of the polyphenols extracts. They were identified based on the retention times of the corresponding
standards.
(a)

(1a) (BA) (1a) (BAc) (1b) (DM) (1b) (DMc) (1c) (V) (1c) (Vc)
Gallic acid 64.563 64.827 53.168 116.329 49.236 58.734
Caftaric acid 14.247 7.002 7.907 4.021
Chlorogenic acid 104.362 96.367 64.453 83.111 65.225 29.6
Epigallocatechin 4.087
Cichoric acid 11.022
Catechin 10.002 9.411 6.068
Caffeic acid 0.539
Epicatechin 2.603 2.093 0.824 1.807 1.16
Sinigrin 1.779 1.939
Coumaric acid 1.777 1.587
Hyperoside 8.83 9.08 4.532 3.453 2.536 20.823
Spiraeoside 3.643
Formononetin 26.208 66.753 6.989 1.253 2.682 4.172
(b)

(2a) (CR) (2a) (CRc) (2b) (OR) (2b) (ORc) (2c) (Z) (2c) (Zc)
Gallic acid 67.565 60.985 259.764 56.526 618.536 555.525
Caftaric acid 15.55 6.848 6.559 49.795 33.45
Chlorogenic acid 76.245 35.456 308.9 67.963 132.459 82.863
Epigallocatechin 35.165
Cicoric acid 21.73 160.473 35.504 117.773
Catechin 22.772
Vanillic acid 11.855 5.87 38.845 6.959
Epicatechin 3.27 4.4 35.573 5.926 13.191 3.373
Quercetin 3,4 diglucoside 1.941 17.595
1-3 dicaffeoylquinic acid 17.311
Sinigrin 5.312 16.668 1.193
Siringaldeid 15.045
Hyperoside 24.541 205.9 27.281 28.773 4.323
Taxifolin 2.48 43.945
Indole3 carboxilic a. 0.526 0.27
Spiraeosid 295.418 10.019
Miricetin 0.218
Genistein 2.09 3.651
Kampherol 6.723 6.045
Formononentin 38.648 32.289 25.736 24.985 4.986 4.063
(c)

(3a) (P) (3a) (Pc) (3b) (S) (3b) (Sc) (3c) (Si) (3c) (Sic)
Gallic acid 301.135 416.078 521.645 292.937 428.594
Caftaric acid 203.988 76.12 145.791 49.122 87.6 39.629
Chlorogenic acid 141.223 86.658 325.264 103.334 237.473 73.644
Epigallocatechin 27.028 12.285
Cicoric acid 137.253 67.065 156.018 79.144 288.118 47.206
Catechin 59.173
Vanillic acid 62.34 38.509 63.736
Caffeic acid 2.488
Epicatechin 21.388 9.815 34.427 8.717 56
Quercetin 3,4 diglucoside 34.809 57.5 4.91
1-3 dicaffeoylquinic acid 82.608
Oxidative Medicine and Cellular Longevity 9

(c) Continued.

(3a) (P) (3a) (Pc) (3b) (S) (3b) (Sc) (3c) (Si) (3c) (Sic)
Sinigrin 47.455 12.978 12.793 154.191
Hyperoside 60.343 21.27 32.073 22.749 146.036 6.567
Rosmarinic acid 36.758 56.964 2.502
Taxifolin 14.038 9.127
Spiraeosid 113.72 39.507
Miricetin 3.355
Dadzein 5.67 8.218
Luteolin 3.095
Quercetin 8.595
Genistein 3.732
Naringenin 0.622
Kampherol 1.98 5.727 5.219
Formononentin 54.108 53.785 21.373 12.951 4.527 3.642
(d)

(4c) (NC) (4c) (NCc) (4b) (NF) (4b) (NFc) (4a) (NA) (4a) (NAc)
Gallic acid 38.359 176.217 74.851 79.488 186.945 83.053
Caftaric acid 90.605 38.329 74.017 26.932 41.02
Chlorogenic acid 334.545 122.52 178.263 94.236 752.809 166.8
Cicoric acid 153.017 54.333
Catechin 7.38
Vanillic acid 29.8
Siringic acid 0.951 1.296 1.887
Epicatechin 14.986 0.871 10.9 1.488 34.482
Quercetin 3,4 diglucoside 45.691 5.526 11.229
Sinigrin 1.936
Coumaric acid 6.177 4.377 14.791 3.047
Ellagic acid 18.083
Siringaldeid 30.8 4.311 113.891 17.72
Hyperoside 34.506 6.68
Taxifolin 13.659 21.782
Indole 3 carboxiladeid 5.245 1.18
Miricetin 1.986
Genistein 9.467
Formononentin 35.941 28.137 39.72 5.096 163.336 65.62
Biochanin-A 4.12

cells using a MTT colorimetric test. After 48 h of treat- illustrated from the EC50 values. However, the ANOVA
ment, growth of cells was significantly inhibited in a dose- analysis on the mean EC50 values obtained for the three cell
dependent manner, and, for each extract, an EC50 value lines did not return significant 𝑝 value (𝑝 > 0.05). The highest
was calculated from dose-response curves of the cell lines. EC50 values were measured in A549 cancer cells, using the
In Table 3, EC50 values on three cell lines are reported, extract of variety Pettilanculo, while the lowest was found in
indicating the concentration of the extracts (expressed as CaCo-2 cells, with CR extracts. Generally, the concentrations
𝜇g equivalent to gallic acid, GAE, as a standard, of total need to inhibit the proliferation of Caco-2 cells which were
polyphenols present in the extract/mL) able to reduce cell 1.06–3.64 folds lower than those of A549 and MCF-7 cells
viability by 50%. The extracts of the nonpigmented varieties without exception. Overall, the extracts of P and Pc showed
(BA, DM, and V) did not cause a significant reduction only moderate inhibitory effect against the three cancer cell
of the cell viability, for each cell line, even at the highest lines, while those of CR and NFc were the most active. In
concentration used; the data are not shown and these varieties addition, the extracts of cooked beans were less efficient than
were not included in Table 5. The EC50 values varied strongly the correspondent crude extracts, except for Pc, NFc, and
among the three cell lines and the different extracts tested, NCc, which showed lower EC50 values than the analogous
indicating a different cellular sensitivity to the extracts, as crude extracts. Likely, in these cases, the thermal processing
10 Oxidative Medicine and Cellular Longevity

Table 5: The antiproliferative activity of the extracts on MCF-7, 4

A549, and Caco-2 cells. EC50 values indicate the concentration
(expressed as 𝜇g equivalent of gallic acid of total polyphenols/mL) 2 B
NA
able to reduce cell viability by 50% (±SD). NF Si OR
NCc S

PCA2 (19.79%)
0 ORc Pc Z
MCF-7 cell line CaCo-2 cell line A549 cell line NAc A
SIc Sc Zc
EC50 EC50 EC50
Sample −2
𝜇g GAE mL−1 𝜇g GAE mL−1 𝜇g GAE mL−1
(±SD) (±SD) (±SD)
−4
CR 29.40 (1.13) 28.52 (4.9) 31.32 (4.2)
CRc 44.17 (2.7) 37.49 (6.37) 68.79 (0.61) −6 C
OR 128.17 (1.17) 58.05 (4.16) 130.83 (2.92)
Orc 135.81 (0.54) 77.62 (2.23) 121.09 (2.84) −8
−4 −2 0 2 4 6 8
Z 185.44 (2.04) 49.85 (6.01) 177.96 (0.61)
PCA1 (64.65%)
Zc 196.70 (1.7) 66.29 (3.11) 273.08 (4.69)
P 333.37 (2.3) 186.18 (14.11) 377.40 (8.34) Figure 3: Principal component analysis (PCA): distribution of 12
raw and 12 cooked bean varieties along principal components 1 (PC1)
Pc 115.33 (1.6) 72.23 (8.72) 252.81 (2.28) and 2 (PC2). using 7 variables. White circles indicate nonpigmented
S 165.57 (0.52) 100.17 (2.36) 293.62 (6.75) raw varieties and white squares, nonpigmented cooked varieties.
Sc 181.36 (0.47) 55.65 (2.9) 213.27 (7.16) Red circles represent red raw cultivars, red squares for red cooked
beans, green circles for speckled raw beans, green squares for
Si 102.60 (0.84) 44.46 (3.73) 122.43 (1.5)
speckled cooked beans, black circles for dark raw beans, and black
Sic 158.61 (2.7) 76.50 (2.39) 88.01 (1.15) squares for dark cooked beans.
NA 67.64 (1.06) 53.75 (1.47) 49.40 (0.55)
NAc 82.57 (0.11) 55.46 (2.02) 55.80 (1.26)
NF 63.75 (1.3) 35.79 (4.39) 54.0 (5.51) flavonoids and the antioxidant and antiproliferative activities.
NFc 33.6 (1.27) 29.4 (3.96) 31.92 (3.08) The highest content of anthocyanins was detected in the
NC 119.02 (1.69) 73.53 (2.36) 118.35 (1.63) extracts of black beans (Table 2). Herein, it is important
NCc 107.16 (1.03) 52.97 (1.87) 49.486 (2.56) to underline that such variable contributed to distinguish
this group B from the others (Figure 3). Group C included
the extracts of nonpigmented beans, which exhibited lower
released compounds with inhibitory capacities. Overall, for phenolic contents and antioxidant activities correlated to
the same cell line, the values of the inhibition obtained with nonsignificant antiproliferative effects.
raw samples were not significantly different with respect to Our analysis suggested that flavonoids and anthocyanins
the cooked counterparts. were accountable for the antioxidant and antiproliferative
effects of these extracts. The most active varieties were those
3.7. Principal Component Analysis. We applied principal belonging to the speckled group. The red varieties were only
component analysis (PCA) to characterize the twelve vari- slightly less active and overlapping with speckled group in
eties of beans according to their biological activity. The PCA. The extracts of dark beans, despite a higher content of
method was successfully used in the treatment of data in anthocyanins, exhibited lower AA values than the speckled,
the study of apples [35] and black beans [36]. In our study, suggesting that the flavonoids were mostly responsible for
seven variables were measured in 24 samples (12 raw and 12 such biological activity.
cooked beans), and data were analysed by PCA (Figure 3).
The cumulative percentage of the total variance explained 4. Conclusions
by the first two components was 84.4%. A bidimensional
plot was designed (Figure 3). The distribution of the varieties Common beans are a key food of the Mediterranean diet,
along PC1 and PC2 showed that samples could be divided representing an important source of proteins, fibres, some
into three main groups: group A, which included red varieties minerals, vitamins, and bioactive compounds. We evaluated
and speckled beans positioned near to the centre of the the antioxidant and antiproliferative effects of polyphenolic
bidimensional plot; group B, which comprised black varieties, extracts obtained from old twelve varieties of endemic bean
and group C, corresponding to exclusively nonpigmented (Phaseolus vulgaris L.), of some rural countries of Southern
varieties. Group A included beans with the highest concen- Italy. Our findings confirm that the speckled, red, and black
trations of phenolic compounds and the best antioxidant coloured varieties of beans are more active than the nonpig-
activity. These varieties differed from the others due to their mented ones. Interestingly, cooking of beans, despite causing
higher content of total polyphenols, in particular, flavonoids, a certain loss of bioactive molecules, did not negatively affect
which were able to influence more effectively both the in marked way their biological properties. This undoubtedly
antioxidant activity and the capability to inhibit the growth of may represent a result of considerable importance, in order
the cancer cells. In fact, we found a positive correlation (𝑟 > to safeguard and promote the old varieties of beans for their
0.736) between the total concentration of polyphenols and important nutraceutical potential, enhancing their market
Oxidative Medicine and Cellular Longevity 11

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