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Cooked Beans or Sprouted Beans?

 

 

 

Effect of cooking and germination on phenolic composition and biological properties of dark beans (Phaseolus vulgaris L.)

 

Abstract

Legumes are the base´s diet in several countries. They hold a high nutritional value, but other properties related to human health are nowadays being studied. The aim of this work was to study the influence of processes (boiling or germination) on the phenolic composition of dark beans (Phaseolus vulgaris L. c.v. Tolosana) and their effect on their antioxidant, neuroprotective and anticancer ability. Phenolic composition of raw and processed dark beans was analysed by HPLC-PAD and HPLC–ESI/MS. The antioxidant activity was evaluated by ORAC. Astrocytes cultures (U-373) have been used to test their neuroprotective effect. Anticancer activities were evaluated on three different cell lines (renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62)) by sulphorhodamine B method. Qualitative and quantitative differences in phenolic composition have been observed between raw and processed dark beans that influence the antioxidant activity, mainly for germinated samples which show a decrease of antioxidant capacity. Although every assayed extracts decreased reactive oxygen species release and exhibited cytotoxicity activities on cancer cell lines, raw beans proved to be the most active in neuroprotective and antitumoral effects; this sample is especially rich in phenolic compounds, mainly anthocyanins. This study further demonstrated that phenolic composition of dark beans is related with cooking process and so with their neuroprotective and anticancer activity; cooking of dark beans improves their digestion and absorption at intestinal level, while maintaining its protective ability on oxidative process at cellular level.

Highlights

► Boiling and germination modify phenolic composition of dark beans. ► Boiling induce a decrease of 68% of anthocyanins. ► Germination increases the concentration of isoflavones and myricetin derivates. ► Raw and processed beans exhibited antioxidant capacity. ► Raw, boiled and germinated dark beans show neuroprotective and anticancer activities.

Keywords

  • Phaseolus;
  • Dark beans;
  • Phenolics;
  • Boiling;
  • Germination;
  • Astrocytes;
  • Neuroprotective;
  • Anticancer

1. Introduction

Beans of Phaseolus vulgaris (common beans) are an important food crop both from economic and nutritional points of view, and are cultivated and consumed worldwide. They are a rich source of protein, complex carbohydrates, dietary fibre and minerals but they also contain biologically active phytochemicals which are important for human health ( Rochfort & Panozzo, 2007). Some authors identified several phenolic compounds (flavones, flavonols, isoflavones and their corresponding glycosylated forms; anthocyanins; monomers and oligomers of flavanols; coumarins and phenolic acids such as hydroxybenzoic and hydroxycinnamic acids) ( Aguilera et al., 2011 and Lin et al., 2008). Their distribution is qualitatively and quantitatively different between the two main parts of the seeds, cotyledon and testa; the testa of these legumes mainly contains proanthocyanidins and anthocyanins ( Aparicio-Fernández et al., 2006). Moreover, this composition varies depending on the variety or colour of the seeds. Dark-coloured beans show the highest content in anthocyanins, which are responsible of seed colour, in addition to the above-mentioned phenolic compounds ( Lin et al., 2008).
Some varieties of beans (P. vulgaris, Vigna sinensis) contain several derivatives from ferulic acid ( Dueñas et al., 2005 and Lin et al., 2008). Recent epidemiological and scientific studies show an inverse association between the risk of chronic human diseases and the consumption of polyphenolic rich diet. Most of the positive effects may be related to the increase of plasma antioxidant capacity and so the protection of cell constituents against oxidative damage; this effect may limit the risk of various degenerative diseases associated with oxidative stress such as cancer, diabetes or neurodegenerative diseases ( Tsao, 2010). Nonetheless, the presence of certain anti-nutritional factors in beans makes it necessary to go through any cooking process before their consumption, just to remove or at least minimize their content. Among the lengthiest these processing method include cooking, germination or fermentation, but processing may vary the quantitative composition of certain components ( Chandrasekara et al., 2012, Dueñas et al., 2009 and López-Amorós et al., 2006).
Thermal processes like boiling are applied to legume seeds as the most common method of processing since humankind began to use them as food. This process mainly results in a decrease in the polyphenol content (Aguilera et al., 2011, Siddhuraju, 2006 and Turkmen et al., 2005).
The process of germination applied to legume seeds is becoming more and more prevalent; it is a natural process to obtain healthy food, easily assimilated, with a high content in water, vitamins and minerals; it also helps to restore intestinal flora. During the germination process in seeds, enzymes begin starch and protein hydrolysis, which makes these nutrients an easier substrate to be digested by human body. The germination process generally improves the nutritional quality of legumes, not only by decreasing the anti-nutritional components, but also by increasing the levels of free amino acids, affordable carbohydrates, dietary fibre, as well as the seeds functionality due to the increase in bioactive compounds (Fernández et al., 2006 and López-Amorós et al., 2006); these changes in their composition varied depending of time of germination and it was observed increase or decrease of the bioactive compounds in function of days of germination (Randhir, Kwon, & Shetty, 2007). The changes occurring to the legume matrix during germination may be reflected in their biological properties, and very little study has been done on this subject up to now.
Neurodegenerative diseases are nowadays a health problem of high prevalence, especially in the elderly population (Gandhi & Abramov, 2012). Brain injury leads to the development of oxidative stress and inflammation that ends in neuronal death. Since the antioxidant capacity of neurons is limited they need the support of astrocytes to fight oxidative stress negative consequences (Shih et al., 2003). Astrocytes play an important role in maintaining brain health and prevention of conditions related to oxidation and can therefore be considered an interesting target to study neuroprotective activity of natural products (Barreto et al., 2011 and Maragakis and Rothstein, 2006). On the other hand, several recent studies tend to establish a relationship between beans consumption with a decrease in the incidence of different digestive tumors (colon, liver); this effect may be due to the presence of lectins, glucidic and polyphenolic compounds (Aparicio-Fernández et al., 2008). Moreover Thompson et al. (2012), indicate that dietary common beans (P. vulgaris L.) consumption is associated with reduced mammary cancer risk in human populations and rodent carcinogenesis models.
Taking into account the importance of legumes in the human diet, the aim of this study was to determine the influence of processing on dark beans phenolic composition and the possible relationship between that phenolic content and the antioxidant and neuroprotective and anticancer activities.

2. Materials and methods

2.1. Samples

Dark bean seeds (P. vulgaris L, c.v. Tolosana) were provided by the D.O. Judías de Tolosa (KALITATEA Foundation, Spain). Seeds were cleaned and stored in polyethylene containers at 4 °C until use.

2.2. Chemicals and solvents

Methanol, ethanol and acetonitrile were of HPLC grade. The HPLC grade standard compounds, trans p-coumaric, trans-ferulic and sinapic acids; (+) catechin; hesperetin 7-O-neohesperidoside; naringenin 7-O-rutinoside; naringenin 7-O-neohesperidoside; quercetin 3-O-glucoside; kaempferol 3-O-glucoside; myricetin 3-O-glucoside; apigenin 7-O-glucoside; genistein 7-O-glucoside; daidzein 7-O-glucoside; biochanin A 7-glucoside; biochanin B 7-glucoside and the anthocyanins, malvidin 3-glucoside and cyanidin 3-glucoside, were purchased from Extransynthèse (France).
Phosphate buffered saline (PBS) with and without Ca and Mg, trypsin–ethylene diamine tetra-acetic acid (EDTA) (Invitrogen, Carlsbad, CA, USA), hydrogen peroxide (Sigma–Aldrich, St. Louis, MO, USA); bromide 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT), Triton X-100 and ferrous sulfate heptahydrate from Sigma–Aldrich (St. Louis, MO, USA) were used. Dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), potassium phosphate dibasic anhydrous (K2HPO4), sodium dihydrogen phosphate (Na2HPO4), glucose and ethanol, were purchased from Panreac Química S.A. (Barcelona, Spain). Disodic fluoresceine, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2′-azobis [2-methylpropionamidine] dihydrochloride (AAPH), were provided by Sigma–Aldrich Chemistry (Germany).

2.3. Boiling process

Five hundred gram seeds were soaked in water (3 L) during 16–18 h, at room temperature. The water was then discarded and seeds were boiled at atmospheric pressure for 60 min with water (3 L); seeds were then separated from water, freeze-dried, milled and passed through a 0.5 mm sieve. The obtained flours were stored under vacuum conditions in desiccators at 4 °C and darkness until further analysis.

2.4. Germination process

Two hundred gram seeds were treated with 0.07% sodium hypochlorite (1000 mL) for 30 min. Then, seeds were washed with distilled water until neutral pH and soaked with distilled water (1000 mL) for 5 h and 30 min and shaken every 30 min. The hydrated seeds were placed on a germination tray, where a wet laboratory paper was extended; they were then covered with another wet paper and then introduced into the germination machine (G-120 Snijders International S.L., Holland) where was in contact with the circulating water of the germinator; being the seeds always wet by capillarity. Seeds were germinated in darkness at 20 °C for 7 days. The sprouts were freeze-dried, milled and passed through a 0.5 mm sieve; the obtained flours were stored under vacuum conditions in desiccators at 4 °C and darkness until further analysis.

2.5. Extraction of phenolic compounds

The seed flours obtained from raw and processed beans were extracted with Methanol/Water (80:20 v/v) in an ASE 200 Solvent Extractor (DIONEX Corporation, CA, USA) and the solution evaporated with N2 atmosphere, by a Turbo Vap. evaporator (Zymark Corporation, MA, USA) coupled to the extractor. The extracts were freeze-dried and kept in sealed containers at room temperature protected from light until reconstitution and further analysis.
The dry extracts were dissolved in Methanol/Water (50:50 v/v) for their analysis by high-performance liquid chromatography (HPLC). All samples were filtered through a 0.45 μm cellulose acetate filter (Millipore) before injection; samples were prepared in duplicate.

2.6. Analysis of phenolic compounds

2.6.1. Non anthocyanin compounds

The analysis was carried out on a HPLC-PAD Waters system (Milford. Mass, USA), comprising an autoinjector, a quaternary pump, a photodiode-array detector 2001 and a Nova-Pak C18 (300 × 3.9 mm, 4 μm) column. Analytical conditions were based on those described by Dueñas, Hernández, and Estrella (2006) with slight modifications; mobile phase consisted in two solvents, A: Water/Acetic acid (98:2 v/v) and B: Water/Acetonitrile/Acetic acid (78:20:2 v/v/v) in gradient elution as follows: 100–20% A at 0–55 min; 20–10% A at 55–70 min; 10–5% A at 70–80 min; 100% B at 80–100 min. The flow rate was 1 mL/min from the beginning to minute 55 and 1.2 mL/min from this time to the end of analysis. The column was reequilibrated between injections with 10 mL Acetonitrile and 25 mL of the initial mobile phase. Detection was performed by scanning the absorption between 210 and 400 nm with an acquisition speed of 1 s. A volume of 25 μl was injected. The samples were analysed in duplicate.
Mass spectra were obtained using a Hewlett Packard 1100MS (Palo Alto, CA) chromatograph equipped with an API source and an electrospray ionisation (ESI) interface. The solvent gradient and column employed were identical to those for HPLC-PAD analyses. The ESI conditions were as follows: negative-ion mode of analysis; N2 as the nebulise gas at 275 kPa, drying gas flow rate and temperature of 10 L/min and 340 °C, respectively; voltage at the capillary entrance was set at 4000 V; and variable fragmentation voltage at 100 V (m/z 200–1000) and 250 V (m/z 1000–2500). Mass spectra were recorded from an m/z of 100–2500.

2.6.2. Anthocyanin compounds

The analysis was carried out in the chromatographic system previously described for the analysis of non anthocyanins. The extracts (100 μl) that were previously filtered through a 45 μm membrane were injected onto a Nova Pak C18 (150 mm × 3.9 mm, 4 μm) column at room temperature following the method from Monagas, Nuñez, Bartolomé, and Gómez-Cordovés (2003). Diode-array detection was performed between 260 and 600 nm. Mass spectra analysis was carried out by the method of anthocyanins and in similar HPLC conditions that for non anthocyanin compounds, in positive-ion mode.

2.7. Identification and quantification of the compounds

Chromatographic peaks were identified by comparison of the retention times, UV spectra characteristics and HPLC–ESI/MS, with those of standards. Other compounds, for which standards were not available, were tentatively identified according to their order of elution, UV spectra by HPLC-PAD and data of HPLC–ESI/MS analysis (Dueñas et al., 2009).
Quantification was carried out using the external standard method with commercial standards. The aldaric derivatives of p-coumaric, ferulic and sinapic acids were quantified by the curves of the corresponding free acids; procyanidin dimers and trimers based on the curve of catechin; flavonols, derivatives of myricetin, quercetin and kaempferol by the curves of myricetin 3-O-glucoside, quercetin 3-O-glucoside and kaempferol 3-O-glucoside, respectively. Flavanones, hesperetin and naringenin derivatives, such as hesperetin 7-O-neohesperidoside and naringenin 7-O-neohesperidoside, respectively. Isoflavones quantification was based on the curves from genistein 7-O-glucoside and daidzein 7-O-glucoside.
Quantification of anthocyanins was carried out by peak area measurements at 530 nm with the calibration curves of cyanidin 3-glucoside.
The calibration curves were made by injection of different volumes from the stock solutions over the range of concentration observed for each of the compounds, using a linear regression for the relationship of area sum vs concentration, under the same conditions as for the samples analysed.
The unknown non-flavonoid and flavonoid derivatives were quantified using the calibration curves of the compounds with a similar phenolic chemical structure and corresponding to the same phenolics group.

2.8. Oxygen radical absorbing capacity assay (ORAC)

The determination of ORAC was carried out according to Xu & Chang (2008a), with slight modifications.
The radical scavenging activity of the extracts was determined by the ORAC method using fluorescein as a fluorescence probe. Briefly, the reaction was carried out at 37 °C in 75 mM phosphate buffer (pH 7.4) with fluorescein, 2,2′-azobis [2-methylpropionamidine] dihydrochloride (AAPH) as peroxyl generator and Trolox (1–8 μM) as standard. Twenty microlitres of sample (at different concentrations), blank (phosphate saline buffer) and Trolox calibration solutions were placed on black 96-well untreated microplate and mixed with 200 μl of working fluorescein solution (116.18 nM, in 75 mM phosphate buffer, pH 7.4). AAPH and Trolox standard solutions were prepared daily with phosphate buffer saline (1–8 μM), and fluorescein was diluted from a stock solution. Bean extracts were diluted in Phosphate Saline Buffer (PSB) and Methanol (50:50 v/v). Samples, blank and Trolox were tempered at 37 °C during 10 min; then, 60 μl of peroxyl generator AAPH was added to initiate the oxidation reaction. The plate was automatically shaken every reading and the fluorescence was recorded every minute for 104 min. A Fluostar Optima plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 485-P excitation and 520-P emission filters was used.
Fluorescence measurements were normalised to the curve of the blank. From the normalised curves, the area under the fluorescence decay curve (AUC) was calculated as:
i=98
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AUC=1+∑fi/f0
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where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The net AUC corresponding to a sample was calculated as follows:
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The net AUC was plotted against the antioxidant concentration to obtain the curve regression equation. The ORAC value was obtained by dividing the slope of the latter curve between the slopes of the Trolox curve obtained in the same assay. Final ORAC values were expressed as μmol of Trolox equivalents/mg of legume.
All reaction mixtures were prepared in duplicate, and at least three times independently.

2.9. Assessment of neuroprotective activity

2.9.1. Cell culture and treatments

Studies were performed on cell cultures of astrocytes from human glioblastoma, U-373 cell line, obtained from the ECACC (European Collection of Animal Cell Cultures, Salisbury, Wiltshire, England). Cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM), free from pyruvate (4.5 g/L glucose, l-glutamine and 25 mM HEPES), with 10% foetal bovine serum (FBS) and 0.5% gentamicin (50 mg/mL) in culture flasks 75 cm3, in an 5% CO2 atmosphere at 37 °C. DMEM and supplements were purchased from Invitrogen (Carlsbad, CA, USA). The cell cultures were fed twice a week until confluence.
To perform the biological assessment, extracts were reconstituted with PBS free of calcium and magnesium immediately before use to achieve the final concentrations of 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL, referred to initial flour.

2.9.2. MTT assay for cell viability

MTT reduction assay for cell survival assessment is a putative marker of cellular redox state reflecting mitochondrial activity and is based on the reduction of the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) into a blue formazan product mainly by the mitochondrial enzyme succinate-dehydrogenase (Takahashi, Abe, Gotoh, & Fukuuchi, 2002).
Astrocyte cells were plated at a density of 5 × 104 cells/well. Then, cells were treated with different concentrations of bean (raw and processed) extracts. Following 24 h of incubation at 37 °C in a 5% CO2 atmosphere with 100 μl of DMEM, the medium was aspirated and replaced with 100 μl of MTT (final concentration: 2 mg/mL) and further incubated in darkness for 1 h at 37° C in an oven forward the formation of formazan dark blue crystals. One hundred microlitres of DMSO were then added to each well and the absorbance recorded at a wavelength of 550 nm with a micro-plate reader (Digiscan 340, Assys Hitech GmbH, Austria). Results are expressed as percentage of viability, whereas 100% viability corresponds to the absorbance of untreated cells. Triton X-100 (5%) was used as a negative control.

2.9.3. Assessment of cell injury

Astrocytes were incubated for 24 h in 96-well plates at a density of 5 × 104 cells/well with 100 μl of DMEM. After this, cells were treated with different concentrations of raw and processed bean (boiled and germinated) extracts and incubated for another 24 h. Then, cells were subjected to oxidative stress by the addition of H2O2, FeSO4 and FeSO4 + H2O2 in buffered saline to obtain final dilutions of 1 mM H2O2 and 0.5 mM FeSO4, respectively (IC50 determined earlier). Dilutions of H2O2, FeSO4 and FeSO4 + H2O2 in PBS were freshly made just prior each experiment. Co-incubation was done in an incubator at 37 °C, in a water-saturated atmosphere of 5% CO2 for 30 min. Treatment was then removed and cell injury and cell viability were determined by MTT assays after 24 h as previously described.

2.9.4. Intracellular ROS measurement

Cellular oxidative stress was assessed by monitoring the formation of free radical species using the dichlorofluorescein (DCFH-DA) technique, a photosensitive molecule which is degraded in the presence of light to 2,7-dichlorofluorescein (DCF) (fluorescent compound). The lipophilic DCFH-DA crosses the cell membrane and then undergoes deacetylation. The resulting DCFH is proposed to react with intracellular hydrogen peroxide or other reactive oxygen species to give the fluorescent compound DCF (Wang & Joseph, 1999). The amount of reactive oxygen species (ROS) present in the samples is proportional to fluorescence intensity. Measurements were made with a fluorimeter (FLX-800 Microplate Fluorescence Reader. Bio-Tek Instruments, Inc.) at a wavelength of excitation and emission of 480 nm and 510 nm, respectively.
Astrocytes were plated 72 h before the beginning of the experiment in 96-well plates at a density of 5 × 104 cells/well and incubated for 24 h at 37 °C and 5% CO2 atmosphere. Then, cells were treated with the different concentrations of the extracts and incubated for another 24 h. After removing the medium, cells were incubated with DCFH-DA (0.01 M/DMSO) in PBS glucose (180 mg glucose/100 mL sterile PBS) during 30 min at 37 °C in 5% CO2 atmosphere. After DCFH-DA was removed, cells were washed with PBS glucose (twice) and fluorescence reading was set every 10 min during the first hour and every 15 min during the second one. The excitation filter was set at 485 nm and the emission filter was set at 538 nm.
To assess whether these compounds can protect cells on a hydrogen peroxide-induce oxidative injury model, cells incubated with the beans extracts for 24 h, were afterwards incubated with Fenton reagent (FeSO4 0.5 mM/H2O2 1 mM) for 30 min. Subsequently, Fenton reagent was removed, and cells were incubated with a solution of DCFH-DA as previously. Measures were performed in the fluorimeter every 10 min during the first hour and every 15 min during the second one, at 485 nm excitation and 538 nm emission wavelengths.

2.9.5. Statistical analysis

Each experiment was carried out in triplicate with at least three independent cultures. Values are expressed as mean ± standard deviation (M ± SD) of at least three experiments. The One-way ANOVA followed by a Newman–Keuls’ multiple comparison test was used to compare control and treated groups. p < 0.05 was considered statistically significant. The data of the ORAC method were analysed using a Duncan’s multiple range test.
Analysis of Principal Components was used to relate values of biological activities and phenolic composition, with the statistical package Statistica version 7.1, Statsoft Inc.

2.10. Anticancer activity

Renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62) cell lines were used in these experiments. They were kindly provided by Dr. F. Cortes (Departamento de Biología Celular. Facultad de Biología. Universidad de Sevilla). The human tumour cytotoxicities were determined following protocols established by NCI (Monks et al., 1991). TK-10, MCF-7, UACC-62 cell lines were cultured in RPMI 1640 medium (Bio Whittaker) containing 20% foetal calf serum (FCS), 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. All cell lines were maintained at 37 °C in a 5% CO2 atmosphere with 95% humidity. The cell culture medium was changed twice a week.
The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimised for the toxicity screening of compounds to adherent cells in a 96-well format. Viable cells were counted using a Coulter counter and diluted with medium to give final densities of 15 × 104, 5 × 104 and 100 × 104 cells/mL for TK-10, MCF-7 and UACC-62, respectively. After 24 h, the cells were treated with the serial concentrations of extracts. One hundred microlitres per well of each concentration was added to the plates to obtain final concentration of 250, 25, 2.5, 0.25 and 0.025 μg/mL for the raw (RB), boiled (BB) and germinated (GB) extracts of P. vulgaris L. The final volume in each well was 200 μg/mL. After an incubation period for 48 h, cell monolayers are fixed with 50 μl of cold 50% (w/v) trichloroacetic acid (TAC) and stained for 60 min at 4 °C , after which the excess dye (SRB solution 0.4% w/v in 1% acetic acid) is removed after 30 min incubated, by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 492 nm using a microplate reader. At the end, GI50 values (concentrations required to inhibit cell growth by 50%), TGI (concentration resulting in total growth inhibition) and LC50 (concentration causing 50% of net cell killing) were calculated according to the previously described protocols ( Monks et al., 1991). Two or three experiments were carried out for each extract or compound. The data are given as the mean of three different assays ± S.E.M.

3. Results and discussion

3.1. Phenolic compounds in raw and processed beans

The UV detection provided a complete overview of the main phenolic compounds in every sample. The HPLC–MS technique gave most structural information; analysis of MS spectra recorded for each peak together with MS2, UV and retention times data led to the identification of several compounds from the chromatographic analysis. Table 1 shows the identified phenolic compounds grouped by their different chemical structure.
Table 1. Wavelength of maximum UV spectrum and molecular ions of the identified phenolics in beans.
Compoundsλ max (nm)[M−H]−[M−H]− frag
Non anthocyanins
Galic acid273169
Protocatechuic acid256, 291153
Ferulyl aldaric acid233, 326285209, 193
trans ferulic acid235, 322193
p-Coumaryl aldaric acid233, 316355209, 173
trans p-coumaric acid233, 314173
Sinapyl aldaric acid235, 326415209, 223
Sinapic acid237, 323223
Procyanidin dimer279577425, 289
Procyanidin trimer279865577, 289
Hesperetin 7-neohesperidoside287, 336sh609301
Hesperetin 7-rutinoside288, 336sh609301
Hesperetin 7-glucoside289, 334sh463301
Hesperetin derivative288, 335sh
301
Naringenin 7-neohesperidoside286, 332sh579271
Naringenin 7-rutinoside286, 332sh579271
Naringenin 7-glucoside286, 327sh433271
naringenin-7-methyl ether 2287, 325sh285
Naringenin288, 328sh271
Quercetin 3-rutinoside256, 354609301
Quercetin 3-galactoside257, 361463301
Quercetin 3-glucoside256, 359463301
Quercetin 3-glucoside acetate257, 358505301
Quercetin256, 374301
Kaempferol 3-glucoside264, 348447285
Kaempferol 3-rutinoside acylated262, 347
593, 285
Kaempferol264, 348285
Myricetin 3-glucoside261, 354479317
Myricetin derivative261, 354521317
Myricetin254, 372317
Apigenin 7-glucoside267, 334431269
Biochanin B 7-glucoside259, 305sh429267
Biochanin A 7-glucoside261, 335sh445283
Daidzein derivative261, 298sh
253
Genistein hexose260, 327sh431269
Genistein derivative259
269
trans resveratrol glucoside318389
Anthocyanins
Delphinidin 3-glucoside278, 526465303
Cyanidin 3-glucoside275, 518449287
Pelargonidin 3,5-diglucoside278, 526595271
Pelargonidin 3-glucoside372, 503433271
Delphinidin glucoside acylated274, 526479303
Pelargonidin glucoside acylated278, 334sh, 528447271
Pelargonidin 3-malonylglucoside278, 530519271
Petunidin feruloyl glucose274, 340sh, 526507317
Petunidin derivative274, 528
317
Malvidin derivative271, 530
331
Table options
Quantitative and qualitative differences have been observed in the phenolic composition of raw and processed dark beans (Table 2 and Table 3). The identified compounds (Table 1) were, different aldaric derivatives (galactarics, altraric or glutarics) of p-coumaric, ferulic and sinapic acids ( Lin et al., 2008), which presented λmax in UV spectrum and molecular ions corresponding to these compounds; procyanidin dimers and trimers; flavonols, derivatives of quercetin, myricetin and kaempferol; flavanones, derivatives of naringenin and hesperetin; apigenin derivative; isoflavones, such as genistein and daidzein derivatives, biochanin A and biochanin B derivatives; anthocyanins, derivatives of delphinidin, cyanidin, pelargonidin, petunidin and malvidin ( Table 1). The presence of the above mentioned compounds varies among the samples; the most marked qualitative differences were observed for germinated beans as some isoflavones and myricetin derivatives were identified but not found in raw or boiled beans.
Table 2. Concentration (μg/g) of identified anthocyanins in raw and processed beans.
Compounds (μg/g)RawBoiledGerminated
Pelargonidin 3,5-diglucosideTracesTraces30.46 ± 1.85
Delphinidin 3-glucoside3.55 ± 0.182.31 ± 0.1415.87 ± 1.59
Cyanidin 3-glucoside88.44 ± 3.8728.44 ± 1.069.12 ± 1.05
Pelargonidin 3-glucoside50.72 ± 2.4214.25 ± 1.58nd
Delphinidin 3-glucoside acylated0.11 ± 0.02ndnd
Pelargonidin 3-glucoside acylated0.38 ± 0.02ndnd
Pelargonidin malonylglucoside0.48 ± 0.031.45 ± 0.11nd
Petunidin feruloyl glucosendnd58.76 ± 2.38
Petunidin derivativendnd11.88 ± 1.08
Malvidin derivativendnd17.64 ± 1.67
nd: no detected.
Table options
Table 3. Concentration (μg/g) of phenolic compounds in raw, boiled and germinated beans.
Raw beanμg/gBoiled beanμg/gGerminated beanμg/g
Protocatechuic acid0.21 ± 0.02Protocatechuic acid0.27 ± 0.03Galic acid0.78 ± 0.05
Ferulyl aldaric acid0.62 ± 0.07Ferulyl aldaric acid0.58 ± 0.05Ferulyl aldaric acid2.89 ± 0.17
Ferulyl aldaric acid4.01 ± 0.32Ferulyl aldaric acid0.38 ± 0.03Ferulyl aldaric acid5.78 ± 0.22
Ferulyl aldaric acid1.26 ± 0.11Ferulyl aldaric acid3.92 ± 0.31p-Coumaryl aldaric acid0.45 ± 0.04
Ferulyl aldaric acid8.63 ± 0.34Ferulyl aldaric acid6.07 ± 0.35p-Coumaryl aldaric acid0.16 ± 0.01
Ferulyl aldaric acid20.87 ± 1.07Ferulyl aldaric acid6.32 ± 0.33p-Coumaryl aldaric acid1.11 ± 0.05
Ferulyl aldaric acid10.75 ± 0.52Ferulic acid4.71 ± 0.33p-Coumaryl aldaric acid0.56 ± 0.04
trans ferulic acid7.37 ± 0.26p-Coumaryl aldaric acid0.45 ± 0.03p-Coumaryl aldaric acid1.13 ± 0.03
p-Coumaryl aldaric acid1.49 ± 0.13p-Coumaryl aldaric acid1.54 ± 0.09p-Coumaric acid1.13 ± 0.11
p-Coumaryl aldaric acid1.08 ± 0.07p-Coumaric acid1.59 ± 0.07Sinapyl aldaric acid3.08 ± 0.27
p-Coumaryl aldaric acid3.44 ± 0.32Sinapyl aldaric acid0.91 ± 0.08Hesperetin 7-glucoside1.22 ± 0.09
Sinapyl aldaric acid0.94 ± 0.08Sinapyl aldaric acid3.03 ± 0.20Naringenin 7-glucoside9.18 ± 0.56
Sinapyl aldaric acid6.68 ± 0.29Sinapyl aldaric acid0.76 ± 0.07Quercetin 3-rutinoside8.55 ± 0.61
Sinapyl aldaric acid9.72 ± 042Sinapic acid5.03 ± 0.34Quercetin 3-glucoside acetate11.53 ± 0.41
Sinapic acidtracesProcyanidin dimer0.72 ± 0.06Quercetin0.81 ± 0.06
Procyanidin dimer39.64 ± 1.11Procyanidin dimer1.33 ± 0.05Kaempferol 3-rutinoside acylated3.49 ± 0.17
Procyanidin dimer49.89 ± 1.01Procyanidin dimer5.75 ± 0.31Kaempferol 3-glucoside7.57 ± 0.35
Procyanidin trimer2.72 ± 0.18Procyanidin dimer3.68 ± 0.21Kaempferol9.14 ± 0.64
Procyanidin dimer7.76 ± 0.31Hesperetin 7-neohesperidoside7.56 ± 0.45Myricetin 3-glucoside31.31 ± 1.11
Hesperetin 7-rutinoside12.04 ± 0.58Hesperetin 7-rutinoside1.25 ± 0.12Myricetin derivative8.28 ± 0.43
Hesperetin derivative0.47 ± 0.06Hesperetin derivative0.55 ± 0.04Myricetin derivative14.47 ± 0.87
Naringenin 7-neohesperidoside11.14 ± 0.34Naringenin 7-neohesperidoside0.61 ± 0.07Myricetin3.97 ± 0.18
Naringenin 7-rutinoside3.93 ± 0.31Naringenin 7-rutinoside0.98 ± 0.08Biochanin B 7-glucoside6.09 ± 0.19
Naringenin-7-methyl ether 22.21 ± 0.12Naringenin-7-methyl ether 22.25 ± 0.11Biochanin A 7-glucoside2.31 ± 0.14
Naringenin1.48 ± 0.11Quercetin 3-galactoside6.13 ± 0.32Daidzein derivative0.35 ± 0.03
Quercetin 3-galactoside1.33 ± 0.14Quercetin1.54 ± 0.06Genistein derivative17.11 ± 1.01
Quercetin 3-glucoside8.51 ± 0.57Kaempferol 3-glucoside4.16 ± 0.21Genistein derivative10.15 ± 0.95
Quercetin 3-glucoside acetate9.63 ± 0.41

Genistein hexose0.35 ± 0.02
Quercetin3.41 ± 0.21

Resveratrol glucoside0.94 ± 0.05
Kaempferol 3-glucoside7.42 ± 0.36



Kaempferol1.01 ± 0.01



Apigenin 7-glucoside3.89 ± 0.21



Genistein derivative1.27 ± 0.06



Table options
Some of the anthocyanins have been identified in agreement with data of Lin et al. (2008) (Table 1) and quantified by the specific method for these compounds. Some of the anthocyanins were also detected in the chromatographic development of the non-anthocyanic phenols, when the detection was done at 530 nm.
In raw beans samples, the highest concentration corresponds to anthocyanins (113.72 μg/g), cyanidin-3-glucoside and pelargonidin-3-glucoside being the most abundant (Table 2). After boiling, a decrease of 68% was observed, but cyanidin and pelargonidin glucoside remained as the most abundant. After 7 days of germination, the total concentration of anthocyanins is similar to that of raw beans, but other different anthocyanins were identified and quantified; pelargonidin 3,5-diglucoside, petunidin feruloylglucoside and two compounds tentatively identified such as petunidin and malvidin derivative. The highest concentration corresponds to petunidin-feruloyl-glucoside (58 μg/g) (Table 2). It is remarkable that two methoxylated anthocyanins, petunidin and malvidin, have been detected in bean sprouts but not in raw and boiling beans; the formation of these compounds could be related to a decrease in methoxylated compounds such as ferulic acid and its derivatives, due to the enzymatic activation during germination. These results agree with those published for other legumes (Lin and Lai, 2006 and Lin et al., 2008).
Table 3 shows the quantification of non anthocyanin compounds in the samples grouping them by their chemical structure in order to better compression.
In germinated beans, isoflavones were detected as daidzein, genistein, biochanin A and biochanin B derivatives (Table 1). The total amount being 29.92 μg/g (Table 3); only one genistein derivative (1.27 μg/g) was detected in raw beans. The increase in isoflavones content observed in germinated samples was previously reported for soy, where aglycones quantity augmented in parallel with germination time (Lin & Lai, 2006).
Flavanones were also identified in seeds, mainly hesperetin and naringenin glycosides (Table 1); these compounds content decreased in processed beans from 25.12 μg/g to 13.22 μg/g or 10.41 μg/g after boiling or germination, respectively (Table 3).
Flavonols, such as quercetin and kaempferol as free compounds or derivatives, were found in raw and processed beans (Table 3). Quercetin derivatives content was higher in raw (19.30 μg/g) and germinated (20.08 μg/g) beans than in boiled samples (8.56 μg/g). More differences were found among the kaempferol derivatives, 3.45% and 5.82%, for raw and cooked samples, respectively. After germination, the presence of several derivatives of myricetin together with quercetin and kaempferol derivatives, quercetin 20.89%, kaempferol 14.50% and myricetin 36.56% were detected. It is important to point out the presence of flavonol aglycones, quercetin, kaempferol and myricetin after germination, whereas only quercetin was found in raw and cooked beans.
The highest concentration of non anthocyanin compounds in unprocessed beans corresponds to procyanidin dimers and trimers (100.05 μg/g) (Table 3); this value decreased from 42% in raw beans to 16.1% in boiled beans. After germination, no procyanidins were identified, probably due to the formation of more polymeric procyanidins which were not detected in the analysis conditions but were described in other legumes (López-Amorós et al., 2006).
The analysed beans contained some esterified hydroxycinnamic acids, from ferulic, p-coumaric or sinapic acids which correspond to an aldaric acid (galactaric, altraric or glucaric acid) linked to free acids. The HPLC–ESI/MS analysis for these compounds showed a fragment molecular ion [M−H]− at m/z 209.1 corresponding to the aldaric acid residue, together with the main negative molecular ions ( Table 1). These compounds have been identified as aldaric isomeric forms of the corresponding free hydroxycinnamic acids (ferulic, p-coumaric and sinapic acid), which have been identified in boiled beans; only ferulic and p-coumaric acid were found in raw and germinated beans, respectively ( Table 3).
The aldaric derivatives of ferulic acid (46.14 μg/g) were the most abundant in raw beans (Table 3), followed by derivatives from sinapic (17.34 μg/g) and p-coumaric acids (6.01 μg/g). These compounds have been previously reported in different beans. Dueñas et al. (2005) found aldaric derivatives of ferulic acid being the most abundant compounds in cowpeas (Vigna sinensis). Aguilera et al. (2011) described the presence of these derivatives of ferulic and p-coumaric acids in Pinta and Canellini beans which represented 44% of the phenolics in these beans. Lin et al. (2008) reported the presence of different concentrations of these hydroxycinnamic acids and derivatives in 24 P. vulgaris beans varieties.
After boiling and germination, a decrease in their concentration was observed, although ferulic derivatives remained as the most abundant (Table 3). The percentage distribution for ferulic, p-coumaric and sinapic derivatives was different; in raw and boiled beans, ferulic derivatives represent the highest percentage (19.38% and 24%, respectively) of the total phenolic content, followed by sinapic derivatives (7.28% and 6.59%) and p-coumaric derivatives (2.52% and 2.8%); the most marked decrease corresponds to ferulic and sinapic acids derivatives in germinated beans.
Resveratrol glucoside was only found in germinated beans (0.94 μg/g) and not detected in any other sample.

3.2. Peroxyl radical-scavenging properties of raw and processed beans

Evaluation of the peroxyl radical-scavenging capacity by ORAC assay showed a negative influence of processing, since values were statistically higher for raw samples (0.94 ± 0.05 μmol TE/mg of legume) than for boiled (0.63 ± 0.03 μmol TE/mg of legume) or germinated ones (0.56 ± 0.01 μmol TE/mg of legume). These results agree with those reported for several raw common beans varieties (Aguilera et al., 2011 and Xu and Chang, 2008a) and other legumes (Amarowicz & Pegg, 2008), affected by thermal treatments. In the case of germination the values of antioxidant activity depends of the days of germination, due to the differences in the enzymatic activity along the germination period (Randhir et al., 2007).

3.3. Neuroprotective activity

The assays performed on cell cultures of astrocytes may highlight the possible mechanism of action of the research compounds. All tested concentrations (1 mg/L, 0.5 mg/mL, and 0.25 mg/mL) from raw and processed beans, kept cell viability levels similar to those obtained for untreated cells (Fig. 1). None of the samples exerted a toxic effect on cells by themselves and even, in some cases, they increased cell survival (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL germinated beans and 1 mg/mL boiled beans). However, differences between processes are low, probably due to the fact that the disappearance of some compounds is accompanied by the synthesis of some new ones that may also contribute to the maintenance of cell viability. Thus, López-Amorós (2006) observed the decrease of hydroxybenzoic acid in germinated beans whereas hydroxybenzoic aldehydes content rises; also detected the formation of flavonol glycosides after 4 days germination.
Cell viability in the presence of raw, boiled and germinated beans extracts. ...
Fig. 1.
Cell viability in the presence of raw, boiled and germinated beans extracts. ∗p < 0.05 vs 1% medium.
Figure options
To assess neuroprotective activity of beans, cell viability was evaluated on a hydrogen peroxide (H2O2)-induced rat astrocytes oxidative injury model (Fenton’s reagent at a concentration FeSO4 0.5 mM/H2O2 1 mM, (Gómez-Serranillos et al., 2009). Fig. 2 shows that these compounds exert a statistically significant protective effect although they did not completely revert to the original status.
Cell viability of raw, boiled and germinated beans extract in cellular injury ...
Fig. 2.
Cell viability of raw, boiled and germinated beans extract in cellular injury model (Fenton’s reagent). ∗p < 0.05 vs Fenton’s reagent treated cells.
Figure options
In relation to the prevention of intracellular free radical generation induced by Fenton’s reagent, all samples at every tested concentration significantly reduced free radical time dependent generation (Fig. 3).
Free radical generations by beans extracts. ∗p<0.05.
Fig. 3.
Free radical generations by beans extracts. ∗p < 0.05.
Figure options
In order to establish the relationship between the phenolic composition and neuroprotective properties, an analysis of the Principal Components was carried out on the values of the astrocytes viability or cells viability protection against oxidant toxic and the concentrations of the identified phenolic groups in raw, boiled and germinated dark bean extracts. In the first case, five components were obtained; the first two ones account for the 99.23 % of the total variance (Fig. 4a). In the analysis of phenolics and assessment cell injury in the presence of a toxic (Fig. 4b), other five components were obtain; the first two ones account for the 98.78% of the total variance.
Plot of principal components of the phenolic compounds and viability (a) or ...
Fig. 4.
Plot of principal components of the phenolic compounds and viability (a) or assessment cell injury (b); ORAC values the phenolic compounds (c); anthocyanins and viability vs toxic (d) of raw (+C), boiled (+B) and germinated (+G) dark beans.
Figure options
Fig. 4a shows that viability maintenance was more related to the aldaric derivatives content of ferulic (A-F) and sinapic (A-S) acids, flavanones, procyanidins (Pr) and raw samples. Nevertheless, these values are negatively related to the viability in germinated samples (G); boiled samples (B) hold an intermediate position. As shown in Fig. 4b, protection against an oxidant was more related to boiling process (B) and seems to be independent of the phenolic composition; a negative correlation is obtained with flavonols, quercetin, kaempferol and myricetin derivatives (Q-d, Kf-d, Myr-d).
The analysis of phenolic compounds in raw (C), boiled (B) and germinated beans (G) and the ORAC values led to the obtention of five components; the first two components account for the 98.94% of the total variance (Fig. 4c). The peroxyl radical-scavenging capacity seems to be more related to kaempferol derivatives (K-d) and quercetin derivatives (Q-d) and the germinated beans.
The analysis of anthocyanins in raw (C), boiled (B) and germinated beans (G), the ORAC values and the viability vs toxic ( Fig. 4d), led to three components; the first two ones account for the 97.31% of the total variance. The anthocyanins content (ACNS) is positively related to ORAC value; the viability vs toxic is also related to anthocyanins content.

3.4. Anticancer activity

The data in Table 4 show the cytotoxic effects of the raw (RB), boiled (BB) and germination (GB) extracts of P. vulgaris L. on three different cell lines; TK-10, MCF-7 and UACC-62. The raw (RB) and boiled (BB) extracts exert cytotoxic activity against all experimented cell lines in a concentration-dependent manner at the recommended NCI (USA) doses. The extract of RB was the most cytotoxic on TK-10 line; it caused total growth inhibition (TGI = 57.12) and also produced a 50% of net killing (LC50) at 170.59 μg/mL. The GB extract showed a strong cytotoxic activity measured by all inhibition parameters in breast adenocarcinoma (MCF-7) and melanoma (UACC-62) cell lines; however, no cytotoxic activity against renal adenocarcinoma (TK-10) cells was detected.
Table 4. Phaseolus vulgaris extracts concentrations (μg/mL) required to inhibit cell growth by 50% (GI50), to produce total growth inhibition (TGI) and to cause 50% of net cell killing (LC50).
ExtractsInhibition parametersTK-10MCF-7UACC-62
RBGI5019.12 ± 0.72a31.5 ± 2.22a137 ± 8.66a
TGI57.12 ± 4.12b>250>250
LC50170.59 ± 6.11c>250>250

BBGI5012.95 ± 1.54a0.80 ± 0.01a0.22 ± 0.01a
TGI111.92 ± 7.44b2.66 ± 0.13 NS2.65 ± 0.28 NS
LC50204.15 ± 7.28c8.88 ± 0.21 NS10.82 ± 1.06c

GBGI50>2500.57 ± 0.09a0.07 ± 0.01a
TGI>2502.71 ± 0.23 NS2.72 ± 0.19 NS
LC50>25012.88 ± 1.65 NS96.17 ± 5.28c
The range of doses assayed was 0.025–250 μg/mL. Results are mean ± S.E.M. (n = 3). a, b, c: means significant differences between processing in every cell line. NS: no significant.
Table options
Our results agree with previous studies that demonstrated the antiproliferative activity of some components of beans, different from phenolics compounds, on human breast cancer (MCF7), human hepatoma (HepG2) and nasopharyngeal carcinoma (CNE1 and CNE2) cell lines (Chan, Wong, Fang, Pan, & Ng, 2012). However, phenolic compounds from other vegetables showed antitumoral effects in a great variety of cell lines in vitro and in vivo.

4. Conclusions

Numerous biological activities are attributed to phenolic compounds, many of them related to their antioxidant and free radical scavenger abilities. It has been scientifically proven that an increase in the consumption of these compounds reduces the risk of developing certain illnesses such as pancreatic and colon cancer, neurodegenerative and cardiovascular diseases and inflammation (Amarowicz and Pegg, 2008, Dueñas et al., 2006 and Shahidi and Naczk, 1995). Their antioxidant effects depend not only on their structure, but also on their ability to bind other molecules like proteins, phospholipids, etc. (Bartolomé, Estrella, & Hernández, 2000), which explains the large differences found in the antioxidant ability for these compounds. For flavonoids, the antioxidant activity is attributed to the presence of ortho-phenolic hydroxyl group, catechins being the most powerful antioxidants.
Several studies published on legumes have reported that different processing conditions such boiling of germination may reduce or increase the phenolic content and so their antioxidant properties (Aguilera et al., 2011 and Lin and Lai, 2006). Xu and Chang (2008b) have shown that cooking at high pressure and reduced cooking time preserves the content in antioxidant compounds. It is also highlighted the influence of the solvent used for the extraction, boiling time, temperature, germination period, etc. on phenolic composition (Amarowicz and Pegg, 2008 and Khalil and Mansour, 1995). The smallest reduction in the antioxidant activity in beans is achieved at 121 °C (Ranilla, Genovese, & Lajolo, 2009), as adopted in this work. Moreover, the antioxidants concentration in seeds and their antioxidant activities may also vary depending on the species, cultivar, growing location and environmental conditions.
The present results shows that boiled and germinated Tolosana dark beans keep an adequate level of antioxidant ability on astrocytes, although cooking processes unfavourably affect their antioxidant properties; these results on composition changes agree with those previously reported for other legumes (Lin & Lai, 2006). This fact is especially interesting for germinated seeds as they are more easily digested than the others.
Overall, the results obtained indicated that raw and processed Tolosana dark beans are an important source of natural antioxidant compounds and so may be considered beneficial in the prevention of those diseases in which the production of free oxygen radicals occurs, such as those related to ageing and oxidative stress-related neurodegenerative diseases; as previously describe in other foods (Gopalan et al., 2012 and Kim and Jang, 2011), polyphenolic compounds (mainly kaempferol and quercetin derivatives, together with anthocyanins) may be considered as the main responsible of the antioxidant activity observed. In the analytical methods used, a correlation was obtained between total phenol content and antioxidant activity.

Acknowledgements

This research was supported by the financed project AGL2008-05673-C02-02/ALI. The authors thank Elena González Burgos and María Porres Martínez, for their help in cell cultures operations; the authors also thank Javier Martin Siguero, for his technical assistance.

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      Citing articles (339)
    • Xu and Chang, 2008b
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