Evaluation of the antioxidant and antiproliferative activities of extracted saponins and flavonols from germinated black beans (Phaseolus vulgaris L.)

Food Chemistry 141 (2013) 1497–1503

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Evaluation of the antioxidant and antiproliferative activities of extracted saponins and flavonols from germinated black beans (Phaseolus vulgaris L.) Daniel Guajardo-Flores, Sergio O. Serna-Saldívar, Janet A. Gutiérrez-Uribe ⇑ Centro de Biotecnología-FEMSA, Escuela de Biotecnología y Alimentos, Tecnológico de Monterrey-Campus Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, N.L., Mexico

a r t i c l e

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Article history: Received 11 February 2013 Received in revised form 8 April 2013 Accepted 9 April 2013 Available online 17 April 2013 Keywords: Black beans Antioxidant capacity Antiproliferative activity Germination Principal component analysis

a b s t r a c t Flavonoids and saponins from common beans have been widely studied due to their bioactivity. This research evaluated the effect of germination of black beans (Phaseolus vulgaris L.) on the antioxidant capacity and antiproliferative activity against cancer cell lines of saponins and flavonoids extracted from seed coats, cotyledons and sprouts. Principal component analysis was performed to achieve punctual associations between the black bean saponins and flavonoids concentrations to the antioxidant capacity and the antiproliferative activities. Total phenolic content and antioxidant capacity of extracts were higher when obtained from seed coats, mainly from the 3rd germination day. The extracts obtained from seed coats after 3 and 5 germination days inhibited all cancer cell lines proliferation with no cytotoxicity against control cells. Genistein was related with the activity against mammary cancer cells but flavonols and group B saponins were more related with hepatic and colon cancers. Non-glycosilated flavonols were related with antioxidant capacity. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction In the last decade, several studies have been focused on characterising the phytochemicals and health benefits of common beans (Aparicio-Fernández, Yousef, Loarca-Pina, de Mejia, & Lila, 2005; Gutiérrez-Uribe, Serna-Saldívar, Moreno-Cuevas, HernandezBrenes, & Guajardo-Touche, 2006). Black bean varieties contain higher amounts of bioactive components such as saponins, flavonoids and antioxidant properties compared with other common bean cultivars (Diaz-Batalla, Widholm, Fahey, CastañoTostado, & Paredes-Lopez, 2006; Oomah, Corbé, & Balasubramaniani, 2010). Through the germination process of the beans, the seeds not only transform themselves into a healthier food for human consumption (López et al., 2013), but they also improve their saponin and flavonoid contents (Diaz-Batalla et al., 2006).

Abbreviations: DDMP, 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4one; TPC, total phenolic content; ORAC, oxygen radical absorbance capacity; GAE, gallic acid equivalents; Trolox, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; TE, Trolox equivalents; AAPH, 2,20 -azobis(2-amidinopropane) dihydrochloride; DMSO, dimethyl sulfoxide; PCA, principal component analysis; P1, principal component 1; P2, principal component 2; MCF7, human breast cancer cell; PC3, human prostate cancer cell; HepG2, human liver cancer cell; Caco2, human colon cancer cell; NIH3T3, mouse embryo fibroblast. ⇑ Corresponding author. Tel.: +52 81 8328 4233; fax: +52 81 8328 4262. E-mail address: [email protected] (J.A. Gutiérrez-Uribe). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.04.010

The sprouting modifies the phytochemical content into an interesting mix of antioxidants with health-promoting properties (Guajardo-Flores, García-Patiño, Serna-Guerrero, Gutiérrez-Uribe, & Serna-Saldívar, 2012; Lin & Lai, 2006; Lopez-Amoros, Hernandez, & Estrella, 2006; López et al., 2013). The saponins and flavonoids found in beans have been widely studied due to their protective role against cardiovascular diseases and cancer (Ellington, Berhow, & Singletary, 2005; MacDonald et al., 2005). Saponins have been proven to be valuable antitumor promoters in carcinogenesis due to their antioxidant effect, direct and selective cytotoxic effect against cancer cells and regulation of cell proliferation (Rao & Sung, 1995), especially if combined with certain flavonoids (Konoshima, Kazuka, Haruna, & Ito, 1991). On the other side, black bean extracts rich in flavonoids inhibited the growth of colon, breast, liver and prostate cancer cells via apoptosis without interfering with the proliferation of normal human fibroblast (Bawadi, 2005; Bobe et al., 2008; Hangen & Bennik, 2002; Thompson et al., 2012). The biological activity of saponins and flavonoids depends on the chemical structures as well as the number of contaminants within the extract (Hou et al., 2004; Kaiser, Pavei, & Ortega, 2010). Principal components analysis (PCA) has been used to target the phenolic content and antioxidant activities to specific metabolites throughout different process. PCA was previously used to correlate that chlorogenic acid and its derivatives were responsible of the

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Fig. 1. Effect of soaking and germination on the extract yield of sprouts (A), cotyledons (B), and seed coats (C). Values represent the mean of 3 replicates ± the standard deviation. Different letters in each graph indicate significant differences, by the Tukey test (p < 0.05). ⁄Dry bean tissue is not sprout but hilum.

antioxidant capacity of potatoes during the cooking process (Xu, Li, Lu, Beta, & Hydamaka, 2009). The antioxidant effects of fermented soybean products were correlated to soyasaponins and isoflavonoid derivatives and clearly distinguished during their fermentation time (Kim et al., 2011). The structural relationships of isolated triterpenic saponins were studied using the PCA of the different groups linked to the aglycones (Kaiser et al., 2010). Particularly in common beans, the relationship between astrocytes viability or cell viability protection against oxidants and concentrations of the phenolics groups in raw, boiled and germinated dark bean extracts was carried out using PCA (López et al., 2013). This experiment concluded that flavonols, quercetin, kaempferol and myricetin derivatives were strongly correlated to cell viability and other phenolic compounds such as flavanones, anthocyanins and phenolic acids were not. Previous strategies evaluated the antiproliferative effect of each purified compound isolated from the black bean seed coats towards different cancer cells lines (Dong, He, & Liu, 2007). However, it was concluded that the complex mix of phytochemicals in beans affects the antioxidant and antiproliferative activities. For this reason, it is important to further study the synergistic effects of black bean saponins and flavonoids responsible for the noteworthy antioxidant and anticancer activities.

Anticancer activity of beans has been previously characterised based on the prebiotic effect of polysaccharides (Campos-Vega, Guevara-Gonzalez, Guevara-Olvera, Oomah, & Loarca-Piña, 2010; Cruz-Bravo et al., 2011; Vergara-Castañeda et al., 2012) but to our knowledge, there is lack of studies about the synergistic effects of different types of saponins and flavonoids associated to black bean extracts. Therefore, the aim of this research was to evaluate the effect of germination on the antioxidant capacity and on the antiproliferative activities against breast cancer (MCF7), prostate cancer (PC3), colon cancer (Caco2) and hepatic cancer (HepG2) cell lines. Further, PCA was performed to achieve punctual associations between the black bean saponins and flavonoids to the antioxidant capacity and the antiproliferative activities.

2. Materials and methods 2.1. Extraction of bioactive constituents of black bean The black bean variety used for the experiments was ‘‘San Luis’’ collected from a local distributor. Extractions were carried out with individual lots of seeds as described by Guajardo-Flores et al. (2012). Briefly, each lot was first soaked in distilled water (1:3,

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w/v) for 24 h with aeration at 500 ml/min provided by an aquatic pump. Then, the soaking water was discarded and the resulting wet seeds placed on germination trays in a dark chamber set at 20 °C and 92% relative humidity for 5 days. Samples were taken daily and immediately dehydrated in an oven set at 60 °C for 4 h including the non-germinated raw and soaked samples. All samples were physically separated into sprouts, seed coats and cotyledons. Each anatomical part was weighed, ground into a powder (Coffee and Spice Grinder Krups GX4100, DF, Mexico) and stored in a freezer at 80 °C until analysis. Extraction of phytochemicals was performed according to Guajardo-Flores et al. (2012). Briefly, after homogenisation, 2.5 g of each sample was accurately weighed and extracted with 25 ml of 80% aqueous methanol (DEQ Monterrey, Mexico) and eight 5 mm glass beads (KIMAX, Vineland, NJ) during 30 min at 25 °C in a vortemp (Vortemp 1550, Labnet International, Inc., Edison, NJ). Then, the resulting extract was filtered using a no. 1 Whatman paper and evaporated under vacuum at 60 °C (Speedvac concentrator, Savant SC210A, Thermo electron Co., Milford, MA) to eliminate the methanol and then freeze-dried (Virtis freezemobile Sentry 2.0, Gardiner, NY). The concentrations of compounds within the extracts were calculated as mg/g of extract for each analysis in order to determine the effect of each compound concentration on each variable tested. 2.2. Determination of total phenolics The total phenolic content of extracts was determined using plates of 96-wells prepared with 15 ll of the Folin–Ciocalteu phenol reagent from Sigma (St. Louis, MO) diluted 1:8 in distillated water and 15 ll of the methanolic extract with 240 ll of distillated water were added. The mixture was allowed to stand for 3 min with gentle shaking, and then 30 ll of sodium bicarbonate 1 N was added. The absorbance of the resulting solution was measured at 750 nm after 2 h. Gallic acid was used as standard and total phenols were calculated as mg gallic acid equivalent (GAE)/g of extract. 2.3. ORAC-FL assay The ORAC method was performed according to Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, (2002). A Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) was used with fluorescence filters set to an excitation wavelength of 485 ± 20 nm and an emission wavelength of 530 ± 25 nm. The plate reader was controlled by software KC4 (Bio-Tek Instruments, Inc., Winooski, VT). The 96-well polystyrene microplate and the covers were purchased from Corning (Corning, NY). Trolox from Aldrich (Milwaukee, WI) was used as standard and antioxidant activities were calculated as microtrolox equivalent (lmol TE)/g of extract. 2.4. Identification and quantification of saponins, flavonols and isoflavones Saponins and flavonoids in methanolic extracts were quantified using an HPLC-DAD-ELSD (Agilent Technologies, Santa Clara, CA) system according to Guajardo-Flores et al. (2012). The extracts were resuspended in 80% methanol to a concentration of 1 mg/ ml prior their injection to the HPLC. Elution was conducted with (A) HPLC-grade water adjusted to pH 2 with trifluoroacetic acid (Sigma, St. Louis, MO) and (B) HPLC-grade acetonitrile. Separation was achieved with 20% B for the first 6 min, increasing the B concentration to 50% at 12 min and to 100% at 30 min. Peak identification of flavonoids was based on retention time and UV spectra. Likewise, flavonoids were quantified using standard curves from

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authentic standards of aglycones. All saponins were quantified using the evaporate light scattering detector (ELSD) and calculated by using a standard curve obtained from soyasaponin I purified in our laboratory (Guajardo-Flores et al., 2012). To confirm the detection of the 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4one (DDMP) conjugated saponins their UVmax absorption maximum at 295 nm was obtained. 2.5. Measurement of inhibition activities against tumor cell proliferation Human hormone-dependent mammary (MCF7) liver (HepG2), colon (Caco2) and prostate (PC3) cancer cells were maintained separately in DMEM-F12 medium containing 10% Fetal Bovine Serum (Gibco, Grand Island, NY). A non-transformed cell line, mouse embryo fibroblasts (NIH3T3), was used to study the specificity of extracts towards cancer cell proliferation. All cells were grown at 37 °C in a humidified 5% CO2 atmosphere and maintained at a cell concentration between 8  104 and 1  105 cells/cm2. Plates of 96-wells were prepared with 100 ll of a suspension containing 5  104 cells/ml of each cell line at least 12 h before adding the extracts, previously resuspended in dimethyl sulfoxide (DMSO). All extracts were tested at a final concentration of 1 mg/ml. After 48 h incubation at 37 °C in a humidified 5% CO2 atmosphere, 20 ll of CellTiter 96Ò AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) were used to determine cell viability by measuring the absorbance at 490 nm in a microplate reader (Synergy HT, Bio-Tek, Winooski, VM). The cell growth inhibition was calculated with the following formula:

100 ðAbsorbance units ðAUÞ of untreated cells  AU treated cellsÞ=AU untreated cells: 2.6. Statistical analysis Statistical analyses were conducted by one-way ANOVA, and differences among means were compared with Tukey’s tests with a level of significance of p < 0.05 using the JMPÒ Version 5 software (SAS Institute Inc., Cary, NC, USA). All analyses were done in triplicate and results were expressed as means ± standard deviations. Principal component analysis was performed to determine deviation of TPC, ORAC and the antiproliferative activities against the various types of cancer cells due to different mass concentrations of saponins and flavonols per g of extract. 3. Results and discussion 3.1. Extraction yield, total phenolics and the antioxidant activities of black bean extracts The highest extraction yield was obtained from germinated sprouts (Fig. 1A). Interestingly, sprouts obtained after soaking had a higher extraction yield (344 mg/g) than the obtained from dry bean hilum (199 mg/g). On the 1st and 2nd germination days the extract yield kept increasing to a maximum of 612 mg/g, suggesting that the complex matrix was transformed into compounds with shorter chain length due to the intrinsic enzyme activity. Further germination of sprouts reduced the extraction yield, but all were higher compared to one-day soaked sprouts. A similar behaviour occurred on cotyledons during germination (Fig. 1B). Soaking did not affect the cotyledons extract yield compared to dry extracts. After the 1st germination day the extract yield increased from 31 to 115 mg/g, reaching its higher extract yield on the 4th day with 315 mg/g extract. A different behaviour was observed in seed coats, in which the soaking process reduced the extraction

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Fig. 2. Effect of soaking and germination on the total phenolic content (TPC) of sprouts (A), cotyledons (B), and seed coats (C). Values represent the mean of 3 replicates ± the standard deviation. Different letters in each graph indicate significant differences, by the Tukey test (p < 0.05). ⁄Dry bean tissue is not sprout but hilum.

yield from 116 to 22 mg/g (Fig. 1C) likely due to the lixiviation of compounds from seed coats into water (Xu & Chang, 2008). After the 1st germination day the extract yield was reduced to only 9 mg/g. The total phenolic content (TPC) of extracts ranged from 0.3 to 0.9 mg GAE/g of extract in sprouts, from 0.3 to 2.2 mg GAE/g of extract in cotyledons and, 27.0 to 61.0 mg GAE/g of extract in seed coats (Fig. 2A–C). Sprouts and cotyledons had a lower total phenolics compared to seed coats in concordance to previous studies in beans (Ranilla, Genovese, & Lajolo, 2007). Neither soaking nor germination affected the TPC in sprout and cotyledon extracts. Interestingly, despite that soaking reduced the seed coat extract yield, there was no significant reduction on the TPC of the extract (p < 0.05). Previous studies reported that TPC of black bean seed coat was reduced by soaking (Fig. 2C) (Lopez-Amoros et al., 2006) but extraction yield was not considered and therefore the TPC of the extract was not analysed. Also, during germination the TPC of black bean seed coats extracts increased from 55.5 to 61.0 mg GAE/g extract on the 2nd, 3rd and 4th germination days, with no significant difference among them. The antioxidant capacity (ORAC) ranged from 9.7 to 27.0 lmol TE/g extract in sprouts, 11.1 to 95.8 lmol TE/g extract in cotyledons and, 132.4–580.0 lmol TE/g extract in seed coats (Fig. 3A–C). Neither soaking nor germination affected ORAC in black bean sprouts extracts. However, germination reduced the ORAC values in cotyledons from 95.8 to 27.7 lmol TE/g extract, respectively (Fig. 3B). The dry and soaked cotyledons were

adequate sources of antioxidant compounds which were not necessary related to phenolics. However, the metabolites produced in cotyledons during germination reduced the antioxidant capacity. Overall, ORAC values of black bean were concentrated on seed coat as previously reported and were correlated with the TPC (p < 0.05) (Oomah et al., 2010; Xu & Chang, 2008). Despite no TPC increment was detected, soaking increased the antioxidant capacity on seed coats from 132.4 to 333.6 lmol TE/g (Fig. 3C). In concordance to previous reports, the germination process increased the antioxidant capacity of black beans due to the transformation of phytochemicals associated to seed coats (Lopez-Amoros et al., 2006) by concentrating the content of phenolics in the seed coat extracts (Guajardo-Flores et al., 2012). On the 3rd germination day the maximum ORAC value was achieved with 580.0 lmol TE/g extract, further germination decreased the antioxidant capacity to 438.9 lmol TE/g extract on the 5th germination day. 3.2. Phytochemical profile and antiproliferative activities of black bean extracts The dry seed coat extract showed high cell inhibitions with specific cytotoxicity towards PC3, HepG2 and Caco2 with 77.7%, 90.1% and 81.2% cell inhibition, respectively (Table 1). The extract bioactivity could be explained due to the presence of 116 mg/g extract of glycosylated flavonols such as myricetin-3-O-glucoside, quercetin4-O-galactoside and kaempferol-3-O-glucoside. Previous studies reported that concentrations between 0.3 and 0.8 mg extract/ml

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Fig. 3. Effect of soaking and germination on the antioxidant capacity (ORAC) of sprouts (A), cotyledons (B), and seed coats (C). Values represent the mean of 3 replicates ± the standard deviation. Different letters in each graph indicate significant differences, by the Tukey test (p < 0.05). ⁄Dry bean tissue is not sprout but hilum.

Table 1 Phytochemicals in black bean seed coat extracts correlated to the antiproliferative activities. Dry

Soaked

Germination number day 1st

Mass concentration (mg/g extract)

Cell growth inhibitionB (%)

Soysaponin Af Deacetyl soyasaponin Af Deacetyl soyasaponin Ae Soyasaponin Ba Soyasaponin Bb Soyasaponin Bd Soyasaponin ag Soyasaponin bg Myricetin-3-O-glucoside A Quercetin-3-O-galactoside Kaempferol-3-O-glucoside Quercetin Kaempferol Genistein MCF7 PC3 HepG2 Caco2 NIH3T3

e

0.65 0.33 NS NS NS NS 0.38c 0.28c 13.34a 102.36a 0.75b NS NS NS 69.4b 77.7a⁄ 90.1a⁄ 81.2a⁄ 73.9b

c

1.09 NS NS 0.27c NS NS 0.57bc 0.26c 11.29b 97.68b 1.00a 0.44c 0.07c NS 64.2b 31.3c 58.7d 54.7c 69.9b

2nd cd

0.98 NS NS 0.31bc NS NS 0.62b NS 9.08c 70.78d 0.68c NS 0.08c NS 50.2c 79.4a⁄ 85.7b⁄ 73.1b 75.5b

3rd e

0.54 NS NS 0.29c NS NS 0.33c NS 7.93d 58.59e NS 1.08b 0.13b NS 81.8a⁄ 64.7b 90.6a⁄ 67.0b 68.2b

4th b

1.45 NS 0.28 0.35b NS NS 0.40c NS 12.35ab 73.65d 0.56d 1.14b 0.17b 0.09b 56.0c⁄ 69.7b⁄ 76.6c⁄ 71.9b⁄ 45.1c

5th a

7.38 0.32 NS 0.43a NS NS 3.14a 0.62a 11.87b 80.88c 0.58d 2.24a 0.30a NS 64.3b 71.4b 75.5c 72.0b 89.1a

0.84d 0.30 0.27 NS 0.29 0.23 0.54bc 0.42b 8.50cd 33.30f NS 2.03a 0.32a 0.18a 79.9a⁄ 41.2c⁄ 39.6d⁄ 38.6d⁄ 30.7d

Different letters in each row indicate significant difference. NS – concentration not significant for the extract viability according to the PCA. A This compound was correlated to the principal component 2. B Inhibition of cellular growth was higher than the observed in positive control cell line (NIH3T3) and is represented by*.

of polyphenols inhibited in vitro more than 74% and 95% of colon and prostate cancer cells respectively, without cytotoxicity

towards normal cells (Seeram, Adams, Hardy, & Heber, 2004; Spanou, Stagos, Aligiannis, & Kouretas, 2010).

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The germination process of black beans significantly changed the composition of seed coat extracts after the 2nd germination day, improving the antiproliferative activities against MCF7 and HepG2 cancer cells (Table 1). The difference between the 1st and 2nd germination day seed coat extracts was the reduction of soyasaponin Af concentration with no bioactivity reported so far. Another difference was the presence of quercetin (1.08mg/g extract), which is an efficient antioxidant agent against cancer cells (Gyo-Nam, Young-In, & Hae-Dong, 2011). Furthermore, the 2nd germination day seed coat extracts showed specific cytotoxicity towards PC3 and Caco2 cancer cell lines with a 64.7% and 67.0%, of cell inhibition, respectively. The 3rd germination day seed coat extract was the only one that exerted >55% of cell inhibition in all cancer cell lines (Table 1). This extract presented a unique mixture of aglycons and glycosylated flavonols, isoflavones and group B soyasaponins Ba and ag. Group B soyasaponins and their DDMP conjugates had been previously reported to inhibit the growth of human colon cancer (Caco2) with no cytotoxic effects on normal cells in a range of concentration of 0.2–1.5 mg/ml (Rao & Sung, 1995; Ban et al., 2007; Ellington et al., 2005). The 5th germination day seed coat extract inhibited the growth of all type of cancer cells, similar to what observed on the 3rd germination day seed coat extract. Interestingly, both seed coat extracts were the only ones with specific cytotoxicity towards hormone-dependent MCF7 cancer cells, probably due to the presence of genistein which ranged from 0.09 to 0.18 mg/g extract. This isoflavone reduced >60% cell proliferation of breast cancer through different mechanism of action in a concentration of 0.03–0.06 mg/ ml (Conklin et al., 2007; MacDonald et al., 2005). The 4th germination day seed coat extract exerted similar cell inhibition percentages, however it lost the specificity probably due to the increase in the concentration of phenolic compounds and soyasaponin Af. Previous studies demonstrated that potent antioxidant extracts rich in phenolic compounds could act as pro-oxidant, negatively affecting cell growth and viability of normal cells if their amount is beyond a critical concentration (Spanou et al., 2010). 3.3. Principal component analysis (PCA) In order to establish the relationship between the values of saponins and flavonoids concentration, TPC, ORAC and antiproliferative activity variables, a PCA was carried out in dry, soaked and germinated black bean extracts. The results indicated that five components were involved; the first two accounted for 83.73% of the total variance (Fig. 4). The principal component 1 (P1) accounted for 70.33% and was more related to the antiproliferative cancer cell activities (Fig. 4). This trend clearly suggests the influence of germination in the bioactivity of anticancer compounds associated to the black bean extracts. Cotyledons and sprouts extracts had a large distance from the centre implying a less degree of relationship to P1 compared to what is observed for seed coats. Principal component 2 (P2) had a strong correlation with the mass concentration, indicating that this particular component was related to the compounds found in higher concentration in black bean extracts. The quercetin 4-O-galactoside was the most abundant compound in the seed coat extracts. In this study, a different distribution of cell viability was also observed depending on the black bean tissue used for extraction. In fact, the compounds related to the anticancer effect were found only in seed coats and were mainly dispersed among the area between P1 and the vectors related with the antiproliferative effect on the different cancer cell lines tested (Fig. 5). Also, P1 had a similar direction towards the TPC and ORAC vectors in agreement with previous studies (López et al., 2013), indicating that P1 could be explaining some of the variability of antioxidant compounds and the correlation between these response variables.

Fig. 4. Chemical compounds in black bean extracts related to principal component 2 (P2). Each dot in the figure represents a compound in the black bean tissue extract (square for seed coat; rumble for sprouts; asterisk for cotyledons).

Phytochemicals found in seed coats obtained after the 2nd, 3rd and 4th germination days were the main responsible of the cytotoxic effect in all human cancer cell lines tested. Based on the distance of each group, the 2nd germination day extracts exhibited the strongest bioactivity against all cancer cell viabilities (Fig. 5). Dry seed coat extracts showed a direction towards the HepG2 and Caco2 suggesting the specificity of these extracts against these cancer cell lines. Meanwhile extracts obtained after 5 germination days had more specificity against MCF7 cells. This study is a strong evidence to support that black bean extract bioactivity is influenced by their component as well as their interactions within the extract. The germination process affected the TPC, ORAC and antiproliferative activities against different human cancer cells due to the influence of this physiological process

Fig. 5. Principal component analysis of chemical compounds present in black bean seed coat extracts related to human breast (MCF7), prostate (PC-3), liver (HepG2) and colon (Caco2) cancer cell lines. Each dot represents a compound in the black bean seed coat extracts.

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