Antioxidant Activity of Faba Bean Extracts

Phenolic compounds were extracted from seeds of 22 cultivars of faba bean ( Vicia faba L.) by using 80% (v/v) aqueous acetone. The total phenolic compound and condensed tannins contents of the extracts and their antioxidant activity were determined using the Folin-Ciocalteu’s phenol reagent, vanillin/HCl method, and ABTS and FRAP assays, respectively. The content of total phenolic compounds ranged from 40.7 to 66.1 mg/g extract and from 326 to 574 mg/100 g seeds. Contents of condensed tannins ranged from 2.40 to 49.9 mg/g extract and from 22.2 (FAB) to 365 mg/100 seeds. The extracts and seeds were characterized by Trolox equivalent antioxidant capacity (TEAC) values ranging from 0.550 (FAB 443) to 1.030 mmol Trolox/g extract (FAB 187) and from 4.85 (FAB 318) to 9.81 mmol Trolox/100 g seeds (FAB 187). Ferric-reducing antioxidant power (FRAP) values varied from 0.595 (FAB 443) to 0.908 mmol Fe 2+ /g extract (FAB 5023) and from 4.61 (FAB 297) to 7.90 mmol Fe 2+ /100 g seeds (FAB 187). The total phenolic content of faba bean extract was correlated with the results of the ABTS (r = 0.864) and FRAP (r = 0.862) assays. The coefficients of correlations between the contents of condensed tannins and ABTS and FRAP results were 0.543 and 0.862. We also noted a correlation between results of ABTS and FRAP assays (r = 0.795).


Introduction
Phenolic compounds of plant origin can inhibit or delay the oxidation of nutrients present in food products. In the human organism, natural antioxidants can protect lipids, proteins, and DNA against reactive oxygen and nitrogen species (ROS, RNS) [1,2]. Results of numerous research groups demonstrate the protective effect of consuming phenolic-rich grains, legumes, oilseeds, fruits, berries, and nuts against several chronic diseases [3][4][5][6][7].
Faba bean (Vicia faba) is a species of Fabaceae family. It is native to South America, North Africa, and southwest and south Asia and is extensively cultivated elsewhere. Similar to other legumes, faba bean seeds contain phenolic compounds [16] including condensed tannins [17][18][19]. The antioxidant potential of faba bean was determined using DPPH, FRAP, and ORAC assays [17,20,21]. Very high antioxidant capacity of Vicia faba sprouts was confirmed by Okumura et al. [22].

Material
Plant material consisted of a collection of 22 faba bean accessions derived from Syria, Morocco, Tunisia, Sudan, Egypt, Yemen, Israel, Georgia, Azerbaijan, Tajikistan, Mongolia, Afghanistan, India, Australia, and Poland ( Table 1). Information about seed coat color and 100 seed weight was provided on the basis of results obtained in a field experiment conducted in Cerekwica (Poland, 51°55′N, 17°21′E).
Description of faba bean seeds is reported in Table 1.

Extraction
Phenolic compounds were extracted from grounded and defatted with hexane faba bean seed by using 80% acetone (v/v) at a solid to solvent ratio of 1:10 (w/v), for 15 min at 50°C [23]. Extraction was carried out in flasks placed in a shaking water bath (Elpan 357, Wrocław, Poland). Acetone from the combined extract was evaporated using a Büchi rotary evaporator. The sample was then freeze-dried.

Determination of total phenolic compound contents
The content of total phenolic compounds in the extracts was determined using the Folin-Ciocalteu's phenol reagent [24]. The results were expressed as (+)-catechin equivalents per g of the extract and per 100 g of seeds.

Determination of condensed tannins content
The content of condensed tannins was determined using a vanillin/HCl colorimetric method [25]. The results obtained were reported as mg catechin equivalent per g extract and 100 g of seeds.

ABTS assay
The Trolox equivalent antioxidant capacity (TEAC) was determined using the method of Re et al. [26]. The results obtained were reported as mmol Trolox equivalents per g extract and 100 g of seeds.

FRAP assay
The ferric-reducing antioxidant power (FRAP) assay was performed as previously described by Benzie and Strain [27]. The results obtained were reported as mmol Fe 2+ equivalents per g extract and 100 g of seeds.

Statistical analysis
The results are reported as a mean value of three determinations ± standard deviation. The Pearson correlation was used to determine the relation between results of total phenolics, condensed tannins, TEAC, and FRAP values. Moreover the principal component analysis (PCA) and hierarchical cluster analysis (HCA) with Ward's method using Euclidean distances were conducted. Statistical and chemometric analyses of data were performed using the Statistica software (Windows software package 8.0).

Content of total phenolics and condensed tannins
The content of total phenolics in the extracts was determined using the Folin-Ciocalteu's phenol reagent. The results were expressed as (+)-catechin equivalents Plant Extracts per g of the extract or 100 g seeds. The results are reported in Tables 2 and 3. The content of total phenolics ranged from 40.7 (FAB 219) to 66.1 mg/g extract (FAB 187) and from 326 (FAB 219) to 574 mg/100 g seeds (FAB 5019).
The content of condensed tannins in the extracts was determined using the vanillin/HCl colorimetric method. The results ranged from 2.40 (FAB 297) to 49.9 mg/g extract (FAB 225) and from 22.2 (FAB 297) to 441 mg/100 seeds (FAB 5019) (Tables 1 and 3).
The results obtained in this research confirm the fact that faba bean is a rich source of phenolic compounds as well as condensed tannins. A lower content of total phenolic compounds was previously reported for extracts of pea [28], white bean [29], broad bean [23,30] lupin [31], and grass pea [32]. A similarly high content of total phenolic compounds was previously reported for red lentil [33], green lentil [34], red bean [35], and adzuki bean [36].
The presence of condensed tannins in faba bean seeds determined with the vanillin method was reported by Amarowicz et al.

Statistical analysis
In our study, for the first time, a correlation was calculated between the content of phenolic compounds in the faba bean extracts and their antioxidant activity. The Antioxidant Activity of Faba Bean Extracts DOI: http://dx.doi.org /10.5772/intechopen.85534 coefficients of correlation between the total phenolic content and the results of the ABTS and FRAP assays were 0.864 and 0.862, respectively (Figure 1). The correlations between the content of condensed tannins and results of ABTS and FRAP assays were weaker and characterized by r = 0.543 and r = 0.528 (Figure 2). A correlation was also observed between the results of both assays (r = 0.795) (Figure 3).
In our previous study, we noted a correlation between the total phenolic content and ABTS and FRAP results determined for red bean (r = 0.997 and 0.997, respectively) [35], white (r = 0.480 and 0.850, respectively) [29], and grass pea (r = 0.881 and 0.781, respectively) [32]. Statistically significant correlations between the content of total phenolics and TEAC as well as between the content of condensed tannins and TEAC (r = 0.857 and 0.787, respectively) were reported for extracts obtained from seeds of faba bean, broad bean, adzuki bean, red bean, pea, red lentil, and green lentil [44].
In the principal component analysis (PCA) (Figure 4), the two first components accounted for 93.6% of the total variability between the faba bean cultivars. The analysis includes the content of total phenolic compounds and condensed tannins in seeds as well as their antioxidant potential determined using ABTS and FRAP methods. A considerable variability in terms of the analyzed traits expressed jointly with the grates Mahalanobis distance was recorded for sample 18 from Afghanistan (FAB 187) and samples 8, 10, and 11 from Sudan (FAB 297), Yemen (FAB 219), and Israel (FAB 6318). The close clustering was observed for seeds from Sudan, Yemen, and Afghanistan and for seeds from Morocco, Sudan, and Tajikistan (sample 4, 7, and 15; FAB 5019, FAB 225, and FAB 354).
The hierarchical cluster analysis ( Figure 5) showed several pairs of faba bean accessions (e.g., FAB 337 and FAB 202, FAB 5023 and FAB 604). Two main clusters were observed. The first contained 16 accessions, whereas the second contained only 6. The presence of similar pairs of faba bean accessions from different countries confirms the limitation of the hierarchical cluster analysis in the discrimination of the geographical origin of samples.

Conclusions
The extracts obtained from faba bean were characterized by a high content of phenolic compounds and condensed tannins. Their antioxidant potential was higher than that reported previously for the extracts of other legume seeds. The content of total phenolics and condensed tannins in the faba bean extracts strongly influenced the antioxidant activity of extracts determined using ABTS and FRAP assays.
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