Phenolic composition and mammary cancer cell inhibition of extracts of whole cowpeas (Vigna unguiculata) and its anatomical parts

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Phenolic composition and mammary cancer cell inhibition of extracts of whole cowpeas (Vigna unguiculata) and its anatomical parts J.A. Gutie´rrez-Uribe, I. Romo-Lopez, S.O. Serna-Saldı´var* Departamento de Biotecnologı´a e Ingenierı´a de Alimentos, Centro de Biotecnologı´a, Tecnologico de Monterrey-Campus Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, N.L., Mexico

A R T I C L E I N F O

A B S T R A C T

Article history:

The phenolic profile and anticancer properties of extracts from whole cowpeas, seed coats

Received 5 July 2010

and cotyledons were determined. Seed coats contained at least 5 and 10 times more free

Received in revised form

and bound phenolics compared to whole seeds (75.6 and 31.7 mg/100 g of free and bound

8 May 2011

phenolics, respectively). Seed coats and cotyledons contained about 50% and 95% of free

Accepted 10 May 2011

phenolics, respectively. The major phenolics associated to seed coats were gallic and pro-

Available online 17 June 2011

tocatechuic acid, whereas in cotyledons p-hydroxybenzoic acid was prevalent. Seed coats contained approximately 10 times more flavonoids compared to whole seeds and cotyle-

Keywords:

dons were practically free of flavonoids. After acid hydrolysis, myricetin, quercetin and

Cowpeas

kaempferol were identified in seed coats. Most of the antioxidant activity determined with

Seed coats

the 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absor-

Cotyledons

bance capacity (ORAC) assays was exerted by free phenolics. The free phenolic extract of

Bound and free phenolics Flavonoids Cancer inhibition Antioxidants

whole seeds at a concentration of approximately 100 mg gallic acid equiv. (GAE)/l inhibited 65% the proliferation of hormone-dependent mammary (MCF-7) cancer cells. Extracts of seed coats or cotyledons also inhibited cell proliferation but to a significantly lesser extent, thus indicating synergistic effects between phenolics and other phytochemicals associated to these anatomical parts.  2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Cowpea (Vigna unguiculata) is one of the most popular legume seeds with an estimated world production of 5.39 million metric tonnes (FAO, 2010). It has similar physical and chemical properties as common beans (Phaseolus vulgaris), except for its fat content. Cowpeas contain around 9–10% fat, whereas common beans have only 0.5–1.5% lipids (Samman, Maldonado, Alfaro, Farfan, & Gutierrez, 1999). These legume seeds are staples in many parts of the world. In an earlier study, Sosulski and Dabrowski (1984) determined the phenolic acid composition of 10 legume species

and found that cowpeas contained the highest concentration of antioxidant compounds. Plant flavonoids, anthocyanins, polyphenols and tannins are a diverse group of phytochemicals that occur in legume seeds and have gained attention due to their antioxidant capacity that benefits human health by preventing oxidative stress (Anderson et al., 1984; Hughes, Ganthavorn, & Wilson-Sanders, 1997). Wu et al. (2004) categorized a wide array of foods in terms of antioxidant capacity and concluded that most legume seeds ranked in the group with the highest activity (>2000 lmol Trolox equiv./serving). Cardador-Martı´nez, Loarca-Pin˜a, and Oomah (2002) determined that common beans possess high antioxidant activity

* Corresponding author: Tel.: +52 81 83284322; fax: +52 81 83284262. E-mail address: [email protected] (S.O. Serna-Saldı´var). 1756-4646/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.05.004

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imparted mainly by phenolics, whereas Seneviratne and Harborne (1992) and Ha et al. (2010) observed that seed coats of all Vigna species, including V. unguiculata, contained antioxidants such as procyanidins and prodelphinidin. Plant phenolics are present in the free, ester, and insoluble bound forms. Methanol soluble free and esterified phenolics are the predominant forms in legume seeds (Krygier, Sosulski, & Hogge, 1982). Cai, Hettiarachchy, and Jalaluddin (2003) concluded that acid hydrolysis during fermentation converts esterified phenolics to simple acids. These authors found that protocatechuic acid present in 17 varieties of V. unguiculata varied from trace amounts to 92.7 mg/100 g after hydrolysis. Protocatechuic acid was identified as the major phenolic acid present in esterified moieties. Other phenolics, such as p-hydroxybenzoic, caffeic, p-coumaric, ferulic, 2,4-dimethoxybenzoic and cinnamic acids were also identified. The total phenolics differed among 17 cowpea varieties. Values ranged from 34.6 to 376.6 mg/100 g indicating large intervarietal differences (Cai et al., 2003). Duen˜as, Fernandez, Herna´ndez, Estrella, and Mun˜oz (2005) identified hydroxybenzoic acids and flavonols such as myricetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin feruloyldiglycoside and another diglycoside of quercetin in cowpeas. The aim of this research was to quantify the antioxidant capacity and amount of phenolics in free and insoluble bound forms associated to whole cowpeas, seed coats and cotyledons. Furthermore, the inhibitory effects of free or bound phenolic extracts were determined using human mammary (hormone-dependent MCF-7) cancer cells.

2.

291

920.85 and 962.09, respectively. Free nitrogen extract was determined by difference (NFE = 100  % moisture  % ash  % protein  % fat  % crude fiber).

2.3.

Extraction of free and bound phenolics

Free and bound phenolics were extracted from cowpeas, seed coats and cotyledons according to the methods described by Sosulski and Dabrowski (1984) and De la Parra, Serna Saldivar, and Liu (2007) with the following slight modifications. The extraction procedure of free phenolics consisted of mixing 10 g of ground seeds, seed coats or cotyledons with 20 ml of 80% chilled ethanol for 10 min. After centrifugation (IEC, CentraMP4R, Needham Heights, MA) at 2500g for 10 min, the supernatant was removed and the same extraction protocol repeated. Supernatants were pooled, evaporated (Speedvac concentrator, Savant SC210A, Thermo Electron Co., Milford, MA) at 45 C to 5 ml and reconstituted with water to a final volume of 10 ml. For the determination of bound phenolics, the pellet obtained after free phenolic extraction was digested with 200 ml 2 M NaOH for 1 h at room temperature with continuous shaking. In order to minimize losses, samples were flushed with nitrogen before hydrolysis. The resulting mixture was acidified to pH 1 with 6 M HCl and then extracted with hexane to remove lipids. The final solution was extracted five times with ethyl acetate. Each ethyl acetate fraction was evaporated to dryness using a Speedvac concentrator (Savant SC210A, Thermo Electron Co., Milford, MA) operating at 65 C. Free and bound phenolic extracts were reconstituted in 10 ml water and stored at 80 C until use.

Materials and methods 2.4.

2.1. Physical properties of cowpeas and separation of its anatomical parts Commercial black colored cowpeas were purchased from a local market located in Merida, Yucatan, Mexico. Cowpeas were hand-cleaned to remove foreign material and splits. Test or volumetric weight and 1000 seed weight were determined. Test weight, expressed as kg/hl, was determined according to method 14-40 of the AACC (2000) using the Winchester Bushel Meter (Seedburo Equipment Co., Chicago, IL) and thousand-seed weight by randomly selecting and weighing 100 seeds and the resulting weight multiplied by 10. Clean cowpeas (5 kg) were decorticated in order to obtain seed coats and cotyledons. The milling procedure consisted of first drying cowpeas at 60 C in a convection oven during 3 h. Upon equilibration at room temperature, the seeds were mechanically decorticated for 6 min in a PRL mill (Nutana Machine Co., Saskatoon, SK, Canada) equipped with a set of carborundum abrasive disks. Seed coats were separated from cotyledons by air aspiration and sifting through a sieve with 2 mm diameter orifices.

2.2.

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Chemical composition of cowpeas

Moisture, ash, protein (N · 6.25), ether extract or fat and crude fiber of whole cowpeas, seed coats and cotyledons were determined following AOAC (1990) procedures 925.1, 923.03, 978.2,

Determination of total phenolics and flavonoids

The total phenolic contents of extracts were determined using the Folin–Ciocalteu method as described by Singleton, Orthofer, and Lamuela-Ravento´s (1999). Gallic acid was used as a standard and total phenols expressed as mg gallic acid equiv. in 100 g (dry weight). Free and bound flavonoids were determined by the colorimetric method described by Zhishen, Mengcheng, and Jianming (1999). Flavonoid concentration was expressed as mg quercetin equiv./100 g.

2.5.

Determination of antioxidant capacity

The antioxidant capacity was determined using the ORAC method as recommended by Huang, Ou, Hampsch-Woodill, Flanagan, and Prior (2002) and the ABTS method according to Pellegrini, Miller, and Rice-Evans (1999).

2.6.

HPLC analysis of phenolics and flavonols

Free and bound phenolics extracts of whole grain, seed coats and cotyledons were analyzed HPLC–PDA (Agilent 1100 Santa Clara, CA) using a Zorbax SB-Aq, 4.6 mm ID · 150 mm (3.5 lm) reverse column. Elution was conducted with water adjusted to pH 2 with trifluoroacetic acid (A) and acetonitrile (B) at a flow rate of 0.5 ml/min. The following gradient was used: 0–8 min with 20–50% B, then at 16 min it increased to 100% B. A post-time of 7 min was used to equilibrate to initial

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conditions. Chromatograms were acquired at 280 nm and integrated by HP-Agilent Software (Chemstation for LC Copyright Agilent Technologies 1990–2003). Five microliters injections were made in each run of free phenolics and 50 ll for bound phenolics. Peak quantification of gallic, protocatechuic, p-hydroxybenzoic, coumaric, and ferulic acid in sample extracts was based on retention times of authentic standards. Peak identification was confirmed comparing the kmax values with those reported by Duen˜as et al. (2005) for the same compounds. For other compounds the UV spectra characteristics were compared to those reported by Duen˜as et al. (2005). To quantify flavonols, the extracts were hydrolyzed in 1 ml of 1.2 M HCl for 20 min at 85 C. Subsequently, a C18 SPE cartridge (Strata C18-E, 100 mg/1 ml, Phenomenex) previously conditioned with 1 ml of methanol and 1 ml of H2O was used to clean the samples. Once the samples were loaded, the cartridges were washed with 25% MetOH and the flavonols sample was recovered with methanol, filtrated and 20 ll were injected to HPLC–PDA. Separation was performed in a Zorbax SB-C18, 3 mm ID · 100 mm (3.5 lm) reverse column. Elution was conducted with water adjusted to pH 2 with formic acid (A) and methanol (B) at a flow rate of 0.4 ml/min with the following gradient: 0–25 min with 40–90% B and 7 min of posttime at the initial conditions. Chromatograms were acquired at 365 nm and integrated by HP-Agilent Software (Chemstation for LC Copyright Agilent Technologies 1990–2003). Peak identification and quantification of myricetin, quercetin and kaempferol was based on retention times and kmax values of authentic standards used for calibration.

2.7.

Mammary cancer cell culture

Human hormone-dependent mammary cancer cells (MCF-7) were maintained in DMEM-F12 medium containing 10% fetal bovine serum (Gibco, Grand Island, NY). 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 by subcultivating in a ratio of 1:3 every week. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was used to determine cell viability. Plates of 96-wells were prepared with 100 ll of a suspension containing 5 · 104 cells/ml of MCF-7 at least 12 h before adding the extracts. Extracts of free or bound phenolics were adjusted with cell growth medium to 376.2 mg GAE/l and 100 ll of these solutions were added to the prepared plates. After 48 h incubation, 20 ll of CellTiter were added and absorbance measured at 490 nm in a microplate reader (Synergy HT, Bio-Tek, Winooski, VM). The concentration needed to inhibit 50% cell growth (IC50) was calculated by testing at least five sample concentrations being the highest the one that contained 188.1 mg GAE/l.

2.8.

Statistical analysis

Total phenolics, flavonoids and HPLC characterizations were done in triplicate and results expressed as mean ± standard deviation. The bioactivities of free and bound phenolics extracts from whole cowpeas, seed coats and cotyledons were statistically compared. Statistical analysis was conducted by

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one-way ANOVA followed by the Tukey’s HSD test at a = 0.05 (JMP 5.0.1.2, SAS Institute Inc., Cary, NC).

3.

Results and discussion

3.1.

Physical properties of cowpeas

Cowpeas had black colored seed coats, a test weight of 83.4 kg/hl and a 1000 seed weight of 136.8 g. The kidney shaped seeds had an average length and width of 8 and 6 mm, respectively. The indigenous cowpea used in this study had lower seed weight than most commercial USA or Canadian types. Mechanically-removed coats constituted 9.86% of the seed weight. Sosulski and Dabrowski (1984) reported that Canadian cowpeas only contained 4.2% seed coats. Other common legume seeds such as soybeans and pinto beans ranged from 6.6% to 9.2% (Kay, 1985).

3.2. Chemical and phenolic composition of whole cowpeas, seed coats and cotyledons The Mexican cowpea contained a similar proximate composition compared to counterparts analyzed by Thangadurai (2004). Whole cowpeas contained 11.6% moisture, 28.2% protein, 10.5% fat, 4.9% crude fiber, 4.2% ash and 40.6% nitrogen free extract. As expected, seed coats contained more crude fiber (12.7%) and ash (12.7%), whereas cotyledons higher quantities of protein (35.2%) and fat (15.2%). Whole cowpeas contained about 70% free and 30% bound phenolics, whereas seed coats contained approximately equal percentages (Table 1). Seed coats had at least 5 and 10 times more free and bound phenols compared to whole seeds. The amount and distribution of free and bound phenolics found in this legume seed considerably differ from cereal grains. Most phenolics associated to cereals are located and bound to the fibrous pericarp cells and in these forms exert most of the antioxidant activity. According to Liyana-Pathirana and Shahidi (2006), Adom, Sorrells, and Liu (2003), Sosulski, Krygier, and Hogge (1982) and Lozovaya et al. (1999) approximately 80% of cereal phenolics are bound to cell wall structures. Particularly in millets, there is a wide variability in the percentage of bound phenolics ranging from 9% to 60% (Chandrasekara & Shahidi, 2010). Studies in canola demonstrated a different distribution of free and bound phenolics. These seeds only contained 6% of bound phenolics (Naczk & Shahidi, 1989). In this study, the quantities of free and bound phenolics were 107.3 and 737.7 mg gallic acid equiv./100 g for whole cowpeas and seed coats, respectively (Table 1). As expected, cotyledons contained the lowest amount of phenolics. Interestingly, nearly all of the phenolics (>95%) were free. This is in accordance to Sosulski and Dabrowski (1984) who found that cotyledons contained only soluble esters forms of phenolics. According to the same authors, seed coats of 10 legume species, including cowpeas, contained more than 3 mg/100 g of p-hydroxybenzoic, protocatechuic, syringic, gallic, coumaric and trans-ferulic acids. In cowpeas, the phenolic acid present in the highest amount was coumaric acid followed by ferulic acid (Sosulski & Dabrowski, 1984). According to Cai et al. (2003), ferulic acid is the main free phenolic only in some

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Table 1 – Free and bound phenolics and flavonoids and antioxidant capacity of whole cowpeas, seed coats and cotyledons.a Whole seed

Seed coats

Cotyledons

a

a

Total phenolics (mg gallic acid equiv./100 g) Free 75.57 ± 2.59 Bound 31.74 ± 2.21

368.05 ± 11.21 369.69 ± 17.41

42.31 ± 2.87 1.44 ± 0.15

Flavonoids (mg quercetin equiv./100 g)a Free Bound

983.83 ± 40.59 747.10 ± 9.60

0.09 ± 0.04 Traces

Antioxidant capacity measured by ORAC (lmol Trolox equiv./g) Free 12.96 ± 0.55 Bound 0.53 ± 0.05

14.91 ± 0.40 0.56 ± 0.04

13.35 ± 0.73 0.59 ± 0.15

Antioxidant capacity measured by ABTS (lmol Trolox equiv./g) Free 4.00 ± 0.57 Bound 0.04 ± 0.00

28.06 ± 0.27 0.04 ± 0.00

11.24 ± 0.78 0.03 ± 0.00

97.50 ± 2.00 75.67 ± 6.89

All values are expressed on dry weight basis. Values are means of three observations.

cowpea varieties and the content goes from traces to 6.2 mg/ 100 g. As depicted in Table 2, the main phenolic acid found in whole seed was ferulic acid followed by coumaric and p-hydroxybenzoic acids. This last phenolic acid was the only one detected in the cotyledons. In seed coats, the main phenolic acid was gallic acid followed by protocatechuic, p-hydroxybenzoic and coumaric acids. In fact, gallic acid was the only phenolic detected in the bound fraction, particularly in the seed coat extract. Seed coats contained approximately 10 times more flavonoids compared to whole seeds, whereas cotyledons were practically devoid of these compounds (Table 1). Chang and Wong (2004) reported the presence of four flavonol glycosides in whole seeds and coats of Vigna sinensis but cotyledons contained less than 10% of the total flavonols. Myricetin,

quercetin and kaempferol as aglycones were only quantified in free phenolic acid extracts of seed coats after hydrolysis (Table 2). There are no previous reports indicating the concentration of bound flavonoids in cowpeas and none of the previously reported flavonols were detected in the bound phenolic acids extract. According to Lin and Lai (2006) legume seeds with dark seed coats are more prone to lose phenolics and flavonoids during soaking and germination probably because they contain higher amounts of free moieties as it is confirmed with the results in Table 2. Lozovaya et al. (1999) demonstrated that hydroxycinnamic acids constitute a significant part (0.01–0.19% of the total dry composition) of compounds associated to cell walls of dicotyledons. Fig. 1 compares chromatograms of free and bound phenolic acids of whole cowpeas and its seed coats and cotyledons.

Table 2 – Free and bound phenolic acids and flavonols content in whole cowpeas, seed coats and cotyledons.a Whole seed

Seed coats

Cotyledons

Free phenolic acids (lg/g) Gallic acid Protocatechuic acid p-Hydroxybenzoic acid Coumaric acid Ferulic acid

ND ND 0.95 ± 0.01 1.25 ± 0.52 26.25 ± 3.47

27.09 ± 4.10 18.97 ± 0.45 5.81 ± 0.97 0.62 ± 0.88 ND

ND ND 5.15 ± 0.50 ND ND

Bound phenolic acids (lg/g)a Gallic acid Protocatechuic acid p-Hydroxybenzoic acid Coumaric acid Ferulic acid

ND ND ND ND ND

5.48 ± 2.42 ND ND ND ND

ND ND ND ND ND

Free flavonols (lg/g)a Myricetin Quercetin Kaempferol

ND 1.361 ± 0.36 ND

2.307 ± 0.79 4.029 ± 0.05 6.096 ± 0.81

ND ND ND

Bound flavonols (lg/g)a Myricetin Quercetin Kaempferol

ND ND ND

ND ND ND

ND ND ND

a

a

All values are expressed on dry weight basis. Values are means of three observations. ND = not detected.

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Fig. 1 – Phenolics profiles of cowpea extracts determined with HPLC–PDA at 280 nm. The upper chromatogram was obtained from free phenolics extracted from whole cowpeas, seed coats and cotyledons and bottom shows the corresponding to bound phenolics. Gallic acid (1), protocatechuic acid (2), p-hydroxybenzoic acid (3), coumaric acid (4), and ferulic acid (5).

Absiscic acid, phaseic acid and some of its metabolites had previously been reported in immature seeds of V. unguiculata (Adesomoju, Okogun, Ekong, & Gaskin, 1980). Hsu (1979) reported that free forms are mainly associated to seed coats. The phenolic p-hydroxybenzoic acid, that has previously been determined in 17 cowpea varieties (Cai et al., 2003), was detected only in its free form in the three samples. Also, Espinosa Alonso, Lygin, Widholm, Valverde, and Paredes Lopez (2006) documented the presence of this phenolic acid in a Mexican collection of 62 wild and weedy common beans. Two other peaks with a lambda maximum of less than 210 nm

that may correspond to triterpenes were detected before the gallic acid peak (peak 1 in Fig. 1). Previously, Noorwala, Mohammad, and Ahma (1995) isolated a triterpenoid saponin from cowpeas that was identified as an aglycone of soyasapogenol B. Bound phenolics were mainly associated to seed coats and gallic acid was identified with the corresponding standards (peak 1 in Fig. 1). However, other peaks were observed in the chromatogram and according to their light absorption spectra some were similar to the flavonol conjugates reported for V. sinensis (Duen˜as et al., 2005) but these compounds were not further characterized.

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The total antioxidant capacity of whole cowpeas was 13.5 lmol TE/g (Table 1), which is lower than the reported by Wu et al. (2004) for black and navy beans which contained 80.4 and 24.7 lmol TE/g, respectively. Although seed coats contained at least 8 times more phenolics than cotyledons, there was not a significant difference (p = 0.24) between ORAC values of these two anatomical parts that constituted 9.9% and 90.1% of the seed weight, respectively. Therefore, constituents other than phenolic acids must be contributing to the observed antioxidant activity of cotyledons. This is particularly interesting due to fat associated to this anatomical part. Thus, the contribution of fats should be analyzed independently in future experiments by inclusion of a defatting step prior to the extraction of free phenolics. More than 96% of the total antioxidant activity of whole cowpeas, seed coats and cotyledons was due to free phenolics. This is contrasting to wheat because most of its antioxidant activity (>82%) is attributed to bound phytochemicals (Adom et al., 2003). Results of the ABTS method for free phenolics were higher compared to values reported by Han (2005) for lentils, chickpeas, yellow peas and green peas, but lower than those for soybeans. The antioxidant activity of bound phenolics was lower than any of the legumes previously reported by Han (2005).

3.4. In vitro mammary cancer cell inhibition by cowpea extracts The inhibition of human mammary hormone-dependent cancer cell growth (MCF-7) by free or bound phenolics is summarized in Fig. 2. All extracts were tested at a phenolic concentration of 188.1 mg GAE/l. Free phenolics were more effective compared to bound counterparts. Interestingly, bound phenolics associated to seed coats or cotyledons did not show significant effects on cell viability and only the bound phenolics of the whole cowpea decreased cell viability to less than 50%. Thus, a synergistic effect between phenolics associated to seed coats and counterparts present in cotyledons might have occurred. Particularly, bound phenolics of seed coats promoted the growth of MCF-7 cells. Although we did not analyze the presence of phytoestrogens, such as

isoflavones and oleanolic acid-derived saponins, they are known to be present at different concentrations in legume seeds. These compounds can promote the growth of hormone-dependent cells in vitro. Soyasapogenol B, a triterpenic saponin, inhibited growth of MDA (estrogen-insensitive mammary cancer cells) cells at a concentration of 10 lM but did not significant affect at any concentration the proliferation of estrogen dependent MCF-7 cells (Rowlands, Berhow, & Badger, 2002). This compound has been previously reported in cowpeas (Noorwala et al., 1995). Free phenolic extracts of whole seeds, seed coats or cotyledons, tested at a dosage of 188.1 mg GAE/l, were equally effective in inhibiting mammary cancer cell growth (Fig. 2). However, the IC50 assay indicated that the free phenolic extract from seed coats significantly reduced cancer cell viability at concentrations higher than 100 mg GAE/l (Fig. 3). In the case of cotyledons, a 30% reduction in viability was observed at a phenolic concentration of 50 mg GAE/l but did not further decrease at augmented concentrations. In contrast, the free phenolics in the whole seed extract reduced cell viability to less than 50% at 100 mg GAE/l. Therefore, free phytochemicals found in seed coats acted synergistically with the ones ¨ nning, and Oredsson (2005) found in cotyledons. Janicke, O

125 100

Viability (%)

3.3. Antioxidant activity of whole cowpeas, seed coats and cotyledons

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75 50 25 0 0

50

100

150

200

Phenolics concentration (mg GAE/L) Cotyledons

Whole seed

Seedcoats

Fig. 3 – Effect of different concentrations of phenolics extracted from whole cowpeas, seed coats or cotyledons on mammary cancer cells (MCF-7) viability.

MCF 7

Cell viability %

200 * 150 * 100 50 0 F Whole grain

B Seed coats

Cotyledons

Fig. 2 – Effects of free (F) or bound (B) phenolic extracts from whole cowpeas, seed coats or cotyledons on mammary cancer cells (MCF-7) viability. The concentration used for all assays was 188.1 mg GAE/l. *Significant difference with the rest of the samples (p < 0.001).

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recently determined that ferulic and p-coumaric acids at a concentration of 1500 lM reduced the number of colon Caco-2 cells to 43–75% after 2–3 days of incubation.

4.

Conclusion

Results indicated that whole cowpeas are a good source of phytochemicals because inhibited in vitro cancer cell growth. Besides the presence of flavonoids and antioxidants, mainly associated to seed coats, components of the cotyledons were necessary to increase the anticancer effects. Antioxidant activity was mainly found in free phenolics and those were more bioactive. Additional research is needed in order to determine and isolate the types of cowpea molecules exerting the high in vitro anticancer activity and the possible existence of positive interactions between or among phytochemicals.

Acknowledgments This research was supported by grants from the Research Chair Fund CAT-005 from ITESM-Campus Monterrey and CONACyT SEP-2004-01-45723.

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