Plant Foods Hum Nutr

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 on farm in marginal areas of Central Italy (Lazio region) were investigated in order to evaluate chemical composition of storage proteins and secondary metabolites fractions. The total protein content showed some differences among landraces; the maximum value was next to 30 g for 100 g of dry weight. The seed storage proteins were screened by polyacrylamide gel electrophoresis (SDS/PAGE): seven landraces exhibited phaseolin patterns type S, one landrace showed a phaseolin pattern type T. A morphological analysis of cotyledon parenchyma performed by scanning

R. Perazzini : D. Leonardi : D. Alesiani : A. Canini (*) Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 1-0133 Rome, Italy e-mail: [email protected] R. Perazzini e-mail: [email protected] D. Leonardi e-mail: [email protected] D. Alesiani e-mail: [email protected] S. Ruggeri Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, Via Ardeatina, 546-00178 Rome, Italy e-mail: [email protected] G. D’Arcangelo Department of Sciences and Chemical Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 1-0133 Rome, Italy e-mail: [email protected]

electron microscopy (SEM) revealed differences in size of starch granules. Moreover the polyphenolic composition was investigated using HPLC-APCI; from the methanol extracts a flavonoid, kaempferol, and a coumarin, 5,7dimethoxycoumarin, were identified. To our knowledge, this is the first time that 5,7-dimethoxycoumarin has been reported in P. vulgaris seeds. Keywords Coumarins . Flavonoids . HPLC-APCI . Phaseolus vulgaris L. . Seed proteins . SEM Abbreviations HPLCHigh performance liquid chromatographyAPCI Atmospheric Pressure Chemical Ionization PHAS Phaseolin RT Retention Time MIN Minutes S Sanilac SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulphate–PolyAcrylamide Gel Electrophoresis SEM Scanning Electron Microscope T Tendergreen TFA Trifluoroacetic Acid TIC Total Ions Chromatogram

Introduction The common bean (Phaseolus vulgaris L.) is the world’s second most important bean after soybeans [1], its dry seeds are consumed largely throughout the world. This food represents a rich and inexpensive source of proteins, carbohydrates, dietary fibers and vitamins to millions of people in developed and developing countries [2].

Plant Foods Hum Nutr

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.

Protein Purification Protein fractions (albumins, globulins, glutelins and prolamins) 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)

Standards of kaempferol (3,4′,5,7-tetrahydroxyflavone), quercetin (3,3′,4′,5,7-pentahydroxyflavone), bergapten (5methoxypsoralen) and 5,7-dimethoxycoumarin were purchased from Sigma-Aldrich (St. Louis, MO). All reagents and solvents used were of analytical or HPLC grade purity.

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.

Total Protein Content

Extraction of Polyphenolics from Seed

Levels of crude protein (N×6.25) were estimated following the procedure described by the AOAC [9].

Three grams sample of ground dry seeds was extracted in a Soxhlet apparatus (90 °C for 21 h) with 200 ml of 70%

Chemicals

Plant Foods Hum Nutr Table 1 Characteristics and total protein content of the investigated landraces Landraces

Growth Seed coat colour habita

proteinb

PHAS Type

‘Atina’s Cannellini’ ‘Fagioli del Purgatorio’ ‘Cocco’ ‘Ciavattoni’ ‘Solfarini’ ‘Verdolini’ ‘Gialli’ ‘Regina’

C B B B B B B C

25.67±0.96 22.95±1.59 21.84±0.27 25.47±1.18 24.41±1.99 21.84±1.39 23.24±0.51 29.18±1.96

S S S S S T S S

a b

White White White White Yellow Light green Brown Brown/black

B Bush, C climbing 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 evaporator (Büchi rotavapor EL130) and then was redissolved in 4 ml of 0.1% TFA. Subsequently the extract was purified with C18 solid-phase extraction cartridge (Supelco, Bellefonte, PA). The cartridge was conditioned with 4 ml of methanol, followed by 4 ml of 0.1% TFA. Then the sample was passed through the cartridge under vacuum, the cartridge was washed with 4 ml of 0.1% TFA and finally the phenolic compounds were eluted with 6 ml of methanol.

min. The mobile phase consisted of two solvents; 0.1% formic acid (A), and methanol (B). The gradient was linear to 5% B in 5 min, 50% B in 45 min, 70% B in 65 min. Identification of individual polyphenolics was carried out using their retention time and mass spectrometric data. Quantification of individual compounds was performed using an internal standard (quercetin or bergapten) with a fixed concentration (100 µg/g) and a five-point regression 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, Milford, USA) coupled with a TSQ 7000 mass spectrometer detector with triple quadrupole (Finnigan, Waltham, MA) supplied with a reversed phase C-18 column (5 µm, 4.6 mm × 150 mm) from Whatman (Brentford, UK). Injection volume was 10 µl, and flow rate was 1.00 ml/ Fig. 1 Concentration of protein fractions in the seeds (g/ 100 g protein)

Results and Discussion Total Protein Content The seed protein content of P. vulgaris is influenced by the environmental conditions in which plant growth and

50 45 40

g/100g protein

Atina’s Cannellini 35

Fagioli del Purgatorio

30

Cocco Ciavattoni

25

Solfarini

20

Verdolini

15

Gialli Regina

10 5 0 Albumin

Globulin

*SD < 10% for all samples.

Glutelin

Prolamin

Glob. / Alb.

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. 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 methods 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

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 correspond 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 5,7dimethoxycoumarin, 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 ‘Verdolini’ with 5,7-dimethoxycoumarin (peak 1, RT 3,72 min) (above) and its APCI mass spectrum (below)

RT: 0,14 - 6,65 SM: 7B NL: 6,76E4 m/z= 205,0208,0 MS fagiolo5

3,71 3,72

100 2,27

95

1

2,29

90 85 80 75

Relative Abundance

70 65 60 55 50 45 40

2,02

35 1,93

30 25

1,88 1,85

20

2,74

3,83

2,75 2,81

15

3,29

3,87

2,95

10 0,18

5

0,53 0,56

1,18

4,12

3,55

1,30

4,49

1,74

4,90 4,92 5,27 5,44

6,06

5,72

6,47

6,51

0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

5,0

4,5

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

Relative Abundance

70 65

206,1

60 55 50 45 278,9

40 35

280,6

30 25

222,7

20

220,9

15

194,1 195,9

10

277,1

281,7 288,2

204,4 219,8

5

226,9 238,3 239,9 241,9 228,9 231,5 243,9

255,2

256,6

274,9 272,7

288,8

299,4 298,0

259,6

0

190

200

210

220

230

240

250

260

270

280

290

300

m/z

Among flavonols, we identified kaempferol in ‘Regina’ with a concentration near to 5 mg/kg (4.878±0.466 mg/kg). Unlike the coumarin, the presence of kaempferol has been detected in different varieties of common bean [27, 28] even if this compound has been found more frequently as glycoside derivatives [29, 30]. Kaempferol is one of the phenolic compounds more studied due its antimutagenic and anticarcinogenic activity both in vitro that in vivo [31] and its concentration in P. vulgaris seeds is very variable, ranging from traces (< 0,2 mg/kg) in Tuscan landraces [27] to 209,4 mg/kg in Mexican cultivated varieties [32]. In a study on 62 wild and weedy Mexican bean collections, one

of the main flavonoids which was found by HPLC analysis was kaempferol, and it has been assumed that the variation in polyphenolic contents was more related to the genotype than to the seed coat color [33]. The presence of this compound is very important for the nutraceutical properties because the flavonols are not much affected by heat treatment [34], so their levels keep constant after cooking. The polyphenolics in general have shown several biological activities such as antioxidant, antimutagenic and/or anticarcinogenic as well as scavenging capacity and inhibition of enzymatic activity. Recently, the polyphenolic contents has been correlated with the biological

Plant Foods Hum Nutr Fig. 5 TIC chromatogram of ‘Regina’ with kaempferol (peak 2, RT 4.52 min; above) and its APCI mass spectrum below

RT: 0,11 - 7,27 SM: 7B NL: 3,63E4 m/z= 285,0287,0 MS fagiolo2

3,61

100

3,62

95 90

3,59

85 80 75

Relative Abundance

70 65 60 55 1,87 2,31

50 45

2,32

2,29

1,86

2,43

40 35

3,68

30 25 1,94

20

2,66 2,67

2 4,50 4,55 4,48

3,14

10 5

3,71

2,80 2,83

15 0,11 0,49 0,47

1,14

1,52

4,63

3,91 4,16

1,74

4,98 5,52 5,50

5,85

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 85 287,7

80 75

Relative Abundance

70 65 60 55 50 45 40 291,7

35

292,0

30

228,3

25

227,5

20 15

195,1

10

207,3 205,7 210,7 204,8 212,8

229,8

220,6

279,2 254,9

239,9 242,2

224,0

256,6 258,1

254,2

230,5 231,0

245,4

260,8

292,7

274,7 272,2

293,9 297,5 298,9

280,5

270,5 267,5

308,8

304,0

5 0 190

200

210

220

230

240

250

260

270

280

290

300

310

m/z

Table 2 Contents of 5,7-dimethoxycoumarin in the investigated landraces Landraces ‘Fagioli del Purgatorio’ ‘Cocco’ ‘Ciavattoni’ ‘Solfarini’ ‘Verdolini’ ‘Gialli’ ‘Regina’

activity of bean methanolic extracts; it has been demonstrated the antimutagenic activity against aflatoxin B1 [35] and the induction of apoptosis on HeLa cells [36].

5,7-Dimethoxycoumarin 0.319±0.023 4.320±0.336 1.296±0.284 0.183±0.009 0.423±0.026 1.296±0.039 0.358±0.032

Data are expressed in mg/kg of fresh weight of seed flour: average value±SD

Conclusions In this report, a preliminary investigation of seed storage proteins in P. vulgaris landrace from Lazio region was carried out. Further studies can be to the basis for conservation and safeguard plans. For example the attribution of European marks should support the survival on farm and promote the knowledge of these landraces to more people. Moreover, further chemical analyses of secondary metabo-

Plant Foods Hum Nutr

lites could be interesting, considering their biological activity described in literature.

17.

Acknowledgment This work was financially supported by PRAL 2003/04 Grant from Regione Lazio.

18.

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