Plant Foods Hum Nutr

DOI 10.1007/s11130-008-0095-7

ORIGINAL PAPER

Characterization of Phaseolus vulgaris

L. Landraces Cultivated in Central Italy
Raffaella Perazzini & Donatella Leonardi &
Stefania Ruggeri & Daniela Alesiani &
Giuseppe D’Arcangelo & Antonella Canini

# Springer Science + Business Media, LLC 2008

Abstract Eight Phaseolus vulgaris L. landraces cultivated electron microscopy (SEM) revealed differences in size of
on farm in marginal areas of Central Italy (Lazio region) starch granules. Moreover the polyphenolic composition
were investigated in order to evaluate chemical composition was investigated using HPLC-APCI; from the methanol
of storage proteins and secondary metabolites fractions. extracts a flavonoid, kaempferol, and a coumarin, 5,7-
The total protein content showed some differences among dimethoxycoumarin, were identified. To our knowledge,
landraces; the maximum value was next to 30 g for 100 g this is the first time that 5,7-dimethoxycoumarin has been
of dry weight. The seed storage proteins were screened by reported in P. vulgaris seeds.
polyacrylamide gel electrophoresis (SDS/PAGE): seven
landraces exhibited phaseolin patterns type S, one landrace Keywords Coumarins . Flavonoids . HPLC-APCI .
showed a phaseolin pattern type T. A morphological Phaseolus vulgaris L. . Seed proteins . SEM
analysis of cotyledon parenchyma performed by scanning
Abbreviations
HPLC- High performance liquid chromatography-
APCI Atmospheric Pressure Chemical Ionization
R. Perazzini : D. Leonardi : D. Alesiani : A. Canini (*)
PHAS Phaseolin
Department of Biology, University of Rome “Tor Vergata”, RT Retention Time
Via della Ricerca Scientifica, MIN Minutes
1-0133 Rome, Italy S Sanilac
e-mail: canini@uniroma2.it
SD Standard Deviation
R. Perazzini SDS-PAGE Sodium Dodecyl Sulphate–PolyAcrylamide
e-mail: perazziniraffaella@libero.it
Gel Electrophoresis
D. Leonardi SEM Scanning Electron Microscope
e-mail: leonardi@uniroma2.it
T Tendergreen
D. Alesiani TFA Trifluoroacetic Acid
e-mail: danielaalesiani@libero.it
TIC Total Ions Chromatogram
S. Ruggeri
Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione,
Via Ardeatina, Introduction
546-00178 Rome, Italy
e-mail: ruggeri@inran.it
The common bean (Phaseolus vulgaris L.) is the world’s
G. D’Arcangelo second most important bean after soybeans [1], its dry
Department of Sciences and Chemical Technologies, seeds are consumed largely throughout the world. This food
University of Rome “Tor Vergata”,
Via della Ricerca Scientifica,
represents a rich and inexpensive source of proteins,
1-0133 Rome, Italy carbohydrates, dietary fibers and vitamins to millions of
e-mail: darcangelo@stc.uniroma2.it people in developed and developing countries [2].

After its introduction from America, about five centuries
ago, there was a quick distribution of seeds in Europe [3].
For a long time natural selection and man’s activities played
an important role on these wild plants, these combined
actions resulted in populations called landraces [4]. The
great changes introduced into agricultural systems after
World War II acted as a powerful leveller and changed the
way food was produced and exchanged [5]; improved
cultivars, selected for their characteristics of stability were
introduced.
In several European countries local populations continue
to survive only in marginal areas, where agriculture is
carried on with traditional farming methods [6]. It’s very
important the collection, characterization and conservation
of these old populations to avoid the loss of unexplored
germplasm [7].
Moreover, today dry beans are receiving increasing
attention as a functional food [8], its consumption has been
linked to reduced risk of cardiovascular disease, diabetes
mellitus, obesity, cancer and diseases of digestive tract. The
physiological effects of dry bean consumption may be due
to the presence of abundant phytochemicals including
polyphenolics which possess both anticarcinogenic and
antioxidant properties [8].
The purpose of the present study was to evaluate the
chemical composition of storage proteins and secondary
metabolites fractions of some common landraces collected
in Central Italy as well as the risk of genetic erosion. The
results can promote the safeguard, by farm conservation, of
these landraces and reveal the economical potential.
Materials and Methods
Samples
Dry seeds of P. vulgaris L. were collected in Lazio (Central
Italy); for each landrace were collected 100 seeds and for
each sample were analyzed three replicates.
Chemicals
Standards of kaempferol (3,4′,5,7-tetrahydroxyflavone),
quercetin (3,3′,4′,5,7-pentahydroxyflavone), bergapten (5-
methoxypsoralen) and 5,7-dimethoxycoumarin were pur-
chased from Sigma-Aldrich (St. Louis, MO). All reagents
and solvents used were of analytical or HPLC grade
purity.
Total Protein Content
Levels of crude protein (N×6.25) were estimated following
the procedure described by the AOAC [9].
Plant Foods Hum Nutr
Protein Purification
Protein fractions (albumins, globulins, glutelins and prola-
mins) were obtained according to Chagas and Santoro [10].
After the coats were removed, the seeds were ground to a
fine meal. The flours (3 g) were defatted overnight with
hexane (1:10 g/ml) at room temperature, washed twice with
ethyl ether for 10 min and dried under an air stream at room
temperature for 1 hour. For each defatted sample 2 grams of
seed flour were extracted by stirring with 20 ml of 0.05 M
borate buffer (pH 7.6) for 2 h and then centrifuged at
30,000 g for 30 min. The supernatants were dialyzed against
a 0.033 M Na-acetate buffer (pH 5.0), for at least 3 days at
4 °C to precipitate the globulins. The pellets were washed
twice with 0.033 M Na-acetate buffer (pH 5.0), suspended in
distilled water and freeze-dried. The supernatants, containing
the albumin fraction, were dialyzed overnight against
distilled water at 4 °C and freeze-dried. Then the albumins
were dissolving in distilled water and centrifuged at 30,000 g
(BECKMAN L7) for 15 min. The purified supernatants were
freeze-dried. The prolamins were solubilized with 70%
ethanol, the glutelins were solubilized with 0.2% NaOH.
Phaseolin was purified by the method of Sathe [11]; this
protein was solubilized with an acid salt solution (0.5 M
NaCl in 0.025 M HCl) at 4 °C and after subsequent
dilutions the final precipitate was dissolved in 0.5 M NaCl,
dialyzed overnight against distilled water and freeze dried.
Electrophoresis
Electrophoresis was performed according to Laemmli [12]
using slab gels of 12.5% acrylamide concentration; the run
was carried out at 200 mV for 1 h. Staining was performed
with Coomassie Brilliant Blue R-250. The molecular weight
markers used were obtained from Biorad (Hercules, CA).
Scanning Electron Microscopy (SEM)
Bean’s cotyledons were sectioned with a razor blade and fixed
for 2 h with 2.5% glutaraldehyde in a 0.1 M phosphate buffer
at pH 7.2, washed three times in the buffer and postfixed in
1% (w/v) osmium overnight at 4 °C [13, 14]. After buffer
washing and dehydration in an ethanol series, samples were
dried to the critical point (BALTEC CPD 030), mounted on
stubs and then gold sputtered (AGAR AUTOMATIC
SPUTTER COATER B7341). The samples were observed
under a scanning electron microscope (Zeiss DSM 950, Carl
Zeiss, Ltd., Montreal, PQ) at 15 kV.
Extraction of Polyphenolics from Seed
Three grams sample of ground dry seeds was extracted in a
Soxhlet apparatus (90 °C for 21 h) with 200 ml of 70%
Plant Foods Hum Nutr

Table 1 Characteristics and total protein content of the investigated

landraces

Landraces Growth Seed coat proteinb PHAS

habita colour Type

‘Atina’s Cannellini’ C White 25.67±0.96 S

‘Fagioli del Purgatorio’ B White 22.95±1.59 S
‘Cocco’ B White 21.84±0.27 S
‘Ciavattoni’ B White 25.47±1.18 S
‘Solfarini’ B Yellow 24.41±1.99 S
‘Verdolini’ B Light green 21.84±1.39 T
‘Gialli’ B Brown 23.24±0.51 S
‘Regina’ C Brown/black 29.18±1.96 S
a
B Bush, C climbing
b
g/100 g of dry seed
Fig. 2 SEM micrograph. Cotyledon parenchyma cells of ‘Cocco’
aqueous methanol (v/v) adjusted to pH 2.0 with HCl. The
methanol extract was evaporated to dryness in a rotary min. The mobile phase consisted of two solvents; 0.1%
evaporator (Büchi rotavapor EL130) and then was redis- formic acid (A), and methanol (B). The gradient was linear
solved in 4 ml of 0.1% TFA. Subsequently the extract was to 5% B in 5 min, 50% B in 45 min, 70% B in 65 min.
purified with C18 solid-phase extraction cartridge (Supelco, Identification of individual polyphenolics was carried
Bellefonte, PA). The cartridge was conditioned with 4 ml of out using their retention time and mass spectrometric data.
methanol, followed by 4 ml of 0.1% TFA. Then the sample Quantification of individual compounds was performed
was passed through the cartridge under vacuum, the cartridge using an internal standard (quercetin or bergapten) with a
was washed with 4 ml of 0.1% TFA and finally the phenolic fixed concentration (100 µg/g) and a five-point regression
compounds were eluted with 6 ml of methanol. curve in the range of 0–20 µg/g on the basis of authentic
standards.
HPLC Analysis

Bean extracts were analyzed on an HPLC system (Waters, Results and Discussion
Milford, USA) coupled with a TSQ 7000 mass spectrom-
eter detector with triple quadrupole (Finnigan, Waltham, Total Protein Content
MA) supplied with a reversed phase C-18 column (5 µm,
4.6 mm × 150 mm) from Whatman (Brentford, UK). The seed protein content of P. vulgaris is influenced by
Injection volume was 10 µl, and flow rate was 1.00 ml/ the environmental conditions in which plant growth and

Fig. 1 Concentration of protein 50

fractions in the seeds (g/ 100 g
protein) 45

40
Atina’s Cannellini
35 Fagioli del Purgatorio
g/100g protein

30 Cocco
Ciavattoni
25
Solfarini
20 Verdolini
15 Gialli
Regina
10

0
Albumin Globulin Glutelin Prolamin Glob. / Alb.
*SD < 10% for all samples.

Fig. 3 SEM micrograph. Cotyledon parenchyma of ‘Solfarini’. Cell
wall and middle lamella residues are visible
seed maturation occur, the genotype of the maternal plant
and the expression of genes that regulate synthesis and
accumulation of protein and nonprotein fractions in the
seed [15].
The total protein amount detected in the eight common
bean landraces is reported in Table 1. Seven of them
showed a protein content varying between 21.8% and
25.8% for 100 g of dry weight; the landrace ‘Regina’ was
characterized by higher value, near to 30%. These results
are in agreement with those reported by other investigations
[13, 15, 16]. The protein content is an important trait to
estimate seed quality and the values obtained, comparable
to those of commercial varieties.
Seed Storage Proteins
Figure 1 shows the amounts of protein fractions extracted
sequentially from the seeds. The albumin contents ranged
from 14.8% to 20.8% while the globulin amounts ranged
from 33.1% to 45.1%. These two fractions represent the
major part of total proteins, together they accounted for
47.9%–65.9%. The glutelin contents ranged from 12.8%
to 41.2%, the prolamins only in three landraces amounted
over 1% of the total protein content. Data from literature
indicate a great disagreement about the amount of legume
protein fractions, often due to different extraction meth-
ods and different recoveries. In the present study, data are
in agreement with the values ranged from 45.5% to
57.2% reported by Chagas and Santoro [10] for globulin
contents. The values obtained for the glutelin amounts
are in one landrace (‘Atina’s Cannellini’) similar to the
average of 10% obtained for American varieties [17].
Phaseolin (PHAS), the major seed storage protein of P.
vulgaris, is considered a biochemical marker [18]. Its
patterns were commonly used to identify gene pools and
the origin of accessions referring to the Mesoamerican or
Plant Foods Hum Nutr
Andean domestication centres [19]. When the protein
extracts were analyzed by one-dimensional SDS-PAGE,
only two PHAS patterns were identified: S (Sanilac) and T
(Tendergreen) as shown in Table 1. ‘Verdolini’ was the only
landrace that showed the T type, typical of the Andean gene
pool; the other landraces showed the S type, typical of the
Mesoamerican gene pool. The last one phaseolin profile is
predominant in this study, but data from literature shows that
the S type is the least diffused in Italy [20], where are
predominant Andean patterns. The frequency of phaseolin
types can changes in different geographical areas [6],
however for explain the origin of germplasm from Lazio
our study can be extended to a larger number of landraces.
SEM Observations
The microstructural characteristics of cotyledon cells are
shown in Figs. 2 and 3. The starch granules, the most
representative storage components, exhibited elliptic or
globular shape and appeared to have different size in
different landraces ranged from about 10×15 µm in ‘Cocco’
(Fig. 2) to 25×25 µm in ‘Verdolini’. These ultrastructural
differences in cotyledon’s parenchyma and cells can corre-
spond to different starch amounts, inclusive the rapidly
digestible fraction.
HPLC Analysis
Bean extracts were subjected to a preliminary analysis for a
qualitative characterization of polyphenolic composition of
the seeds. The compounds were identified by comparison
of their retention times and mass spectra with those of the
standard solutions analyzed under the same conditions. By
the total ions chromatogram (TIC) was identified 5,7-
dimethoxycoumarin, a compound belonging to coumarin
class (Fig. 4). To our knowledge this is the first report on
identification of this compound in P. vulgaris seeds, and in
general in pulse seeds. This coumarin in our study was
identified in seven of the eight landraces investigated; it
wasn’t detected only in ‘Atina’s Cannellini’. By methanol
extracts was also identified a flavonol, the kaempferol,
found only in ‘Regina’ (Fig. 5).
Subsequently was performed a quantitative analysis of
the compounds identified. The higher concentration of the
coumarin was detected in ‘Cocco’ (4,32 mg/kg) and
‘Ciavattoni’ (2,90 mg/kg), both with white seed coat
(Table 2). In literature there are only few data on this
compound that was found in few vegetable species like
Euodia borbonica var. borbonica L. [21], Citrus limon L.
[22], Heracleum mantegazzianum L. [23], Citrus medica
sarcodactylis [24] and Carica papaya [25]. In regard to his
activity on in vitro cultures, an important antiproliferative
activity on B16 cells was demonstrated [26].
Plant Foods Hum Nutr

Fig. 4 TIC chromatogram of RT: 0,14 - 6,65 SM: 7B

‘Verdolini’ with 5,7-dimethoxy- 100 3,71 3,72 NL:
6,76E4
coumarin (peak 1, RT 3,72 95
2,27
1
2,29 m/z=
min) (above) and its APCI mass 90 205,0-
spectrum (below) 85 208,0 MS
fagiolo5
80
75
70
65

Relative Abundance
60
55
50
45
40 2,02
35
1,93
30
25 1,88
3,83
1,85 2,75
20 2,74

15 2,81
3,29 3,87
2,95 4,12
10 3,55 4,49 4,90 4,92
1,30 5,27 5,44 6,06 6,51
0,18 0,53 0,56 1,74 5,72 6,47
5 1,18

0
0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5

Time (min)

fagiolo5#416-461 RT: 3,54-3,92 AV: 46 SM: 7B NL: 1,67E3

T:+ p APCI ms [ 189,94-309,94]
209,3
100
95
90 208,8

85 208,3
80
75
70
65
Relative Abundance

206,1
60
55
50
45
40 278,9

35 280,6
30
25 222,7
20 220,9
277,1 281,7 288,2
15 204,4
194,1 299,4
10 195,9
219,8 226,9 238,3 239,9 241,9 274,9 288,8
228,9 256,6 272,7
231,5 255,2 259,6 298,0
243,9
5
0
190 200 210 220 230 240 250 260 270 280 290 300
m/z

Among flavonols, we identified kaempferol in ‘Regina’ of the main flavonoids which was found by HPLC analysis
with a concentration near to 5 mg/kg (4.878±0.466 mg/kg). was kaempferol, and it has been assumed that the variation
Unlike the coumarin, the presence of kaempferol has been in polyphenolic contents was more related to the genotype
detected in different varieties of common bean [27, 28] than to the seed coat color [33]. The presence of this
even if this compound has been found more frequently as compound is very important for the nutraceutical properties
glycoside derivatives [29, 30]. Kaempferol is one of the because the flavonols are not much affected by heat
phenolic compounds more studied due its antimutagenic treatment [34], so their levels keep constant after cooking.
and anticarcinogenic activity both in vitro that in vivo [31] The polyphenolics in general have shown several
and its concentration in P. vulgaris seeds is very variable, biological activities such as antioxidant, antimutagenic
ranging from traces (< 0,2 mg/kg) in Tuscan landraces [27] and/or anticarcinogenic as well as scavenging capacity
to 209,4 mg/kg in Mexican cultivated varieties [32]. In a and inhibition of enzymatic activity. Recently, the polyphe-
study on 62 wild and weedy Mexican bean collections, one nolic contents has been correlated with the biological
 Plant Foods Hum Nutr

Fig. 5 TIC chromatogram of RT: 0,11 - 7,27 SM: 7B

‘Regina’ with kaempferol (peak 100
3,61
3,62
NL:
3,63E4
2, RT 4.52 min; above) and its 95
m/z=
APCI mass spectrum below 90 3,59 285,0-
85 287,0 MS
80 fagiolo2
75
70
65

Relative Abundance
60
55
2,32
50 1,87 2,31

45 2,29 2,43
1,86
40
35 3,68
30
25
20
1,94
2,66 2,67 3,71
2
4,50
15 2,80 2,83 4,55
4,48
10 3,14
3,91 4,16 4,63
1,74 4,98 5,52 5,50 5,85
1,52
5 0,11 0,49 0,47 1,14 6,30 6,76 7,09

0
0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0
Time (min)

fagiolo2#522-550 RT: 4,43-4,67 AV: 29 SM: 11B NL: 1,03E3

T: + p APCI m s [ 189,94-309,94]
288,5
100
290,0
95
90
85
80 287,7

75
70
65
Relative Abundance

60
55
50
45
40
291,7
35
292,0
30
228,3
25 229,8
227,5 279,2 292,7
20 207,3 256,6 274,7
239,9 242,2 254,9 293,9 297,5
205,7 224,0 258,1 280,5
15 210,7 272,2 308,8
195,1 220,6 230,5 254,2 298,9
204,8 260,8 270,5
10 212,8 231,0 245,4
267,5 304,0
5
0
190 200 210 220 230 240 250 260 270 280 290 300 310
m/z

activity of bean methanolic extracts; it has been demon-

strated the antimutagenic activity against aflatoxin B1 [35]
Table 2 Contents of 5,7-dimethoxycoumarin in the investigated
landraces and the induction of apoptosis on HeLa cells [36].

Landraces 5,7-Dimethoxycoumarin

‘Fagioli del Purgatorio’ 0.319±0.023

Conclusions
‘Cocco’ 4.320±0.336
‘Ciavattoni’ 1.296±0.284 In this report, a preliminary investigation of seed storage
‘Solfarini’ 0.183±0.009 proteins in P. vulgaris landrace from Lazio region was
‘Verdolini’ 0.423±0.026 carried out. Further studies can be to the basis for con-
‘Gialli’ 1.296±0.039 servation and safeguard plans. For example the attribution of
‘Regina’ 0.358±0.032 European marks should support the survival on farm and
Data are expressed in mg/kg of fresh weight of seed flour: average promote the knowledge of these landraces to more people.
value±SD Moreover, further chemical analyses of secondary metabo-
Plant Foods Hum Nutr
lites could be interesting, considering their biological activity
described in literature.
Acknowledgment This work was financially supported by PRAL
2003/04 Grant from Regione Lazio.
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