Σάββατο 21 Νοεμβρίου 2015

Φυτρο Ηλιοσπορο Καλυτερη Αντιοξειδωτικη δραση απο αλλα

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Phenolic profile and antioxidant activity in selected seeds and sprouts

  • Department of Food Analysis and Quality Assessment, Faculty of Food Technology, University of Agriculture, Balicka Str. 122, 30-149 Krakow, Poland
Received 20 March 2013, Revised 5 July 2013, Accepted 16 July 2013, Available online 22 July 2013

doi:10.1016/j.foodchem.2013.07.064
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Highlights

•
We examined the influence of germination on the characteristics of selected seeds and sprouts.
•
Free and bound phenolic compounds, before and after alkaline hydrolysis, were determined.
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Increase of phenolics content and antioxidant activity was observed during germination.
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There were high linear correlations between total polyphenol contents and antioxidant activities.

Abstract

The aim of this study was to investigate the effect of germination on the phenolic acids and flavonoids profile, as well as antioxidant activity (AA), in selected edible seeds of mung beans, radish, broccoli and sunflower. Germination increased the total phenolic (TP) and flavonoid (TF) levels, as well as the AA of the seeds, and influenced the profile of free and bound phenolic compounds. Among the samples, mung bean was characterised by lowest levels of TP and TF, as well as AA, evaluated using ABTS, DPPH and FRAP assays. Sunflower and radish sprouts were the most rich in phenolic compounds. Insignificant amounts of free phenolic acids were found in the free phenolic acid fraction; alkaline hydrolysis of the seeds and sprouts extracts provided the majority of the phenolic acids. The amounts of free and bound flavonoids were inconsiderable both for seeds and sprouts.

Keywords

  • Seeds;
  • Sprouts;
  • Flavonoids;
  • Phenolic acids;
  • Antioxidant activity;
  • HPLC

1. Introduction

Plant-based foods are a good source of nutrients, dietary fibre, mineral and phenolic compounds. Unfortunately, fresh fruits and vegetables are usually seasonal and therefore they are expensive out of season. Furthermore, most of the out-of-season crops are cultivated under artificial conditions, and then they are prematurely harvested and exported to other parts of the world. All these factors result in a decline in a nutrient value of the crops. An excellent alternative for plant foods are sprouts, which can be consumed in fresh form at all times of the year. Edible seeds and sprouts are a good source of antioxidants, such as: phenolic acids, flavonoids, trace elements and vitamins (Pasko et al., 2009). These plants qualify as a functional food, providing natural protection against heart disease and some forms of cancer. Common sprouting seeds include mung beans, radish, broccoli, sunflower, lentil, soybean, alfalfa, cabbage, wheat, rice, pea, amaranth, quinoa and others. In Poland the most popular seeds used for germination are mung bean, radish, broccoli and sunflower.
Germination of edible seeds to produce sprouts increases their nutritive value (Dueñas et al., 2009, Hung et al., 2011 and Martinez-Villaluenga et al., 2010). Several studies have reported higher levels of nutrients and lower contents of antinutrients in sprouts compared to the ungerminated seeds (Martinez-Villaluenga et al., 2010, Oloyo, 2004 and Zieliński et al., 2005). However, information about free and bound phenolics in raw and sprouted seeds is scarce. The content and composition of bioactive compounds in the sprouts depends on many factors, e.g. climatic and agronomic conditions of growth, storage conditions of sprouts, level of their maturity, and also on their variety (Cevallos-Casals & Cisneros-Zevallos, 2010). Due to the fact that sprouts are considered as a cheap and relatively new source of functional foods it is necessary to determine and characterise bioactive compounds occurring in ready-to-eat germinated seeds.
The aim of this study was to investigate the effect of germination on the phenolic acids and flavonoids profile, as well as antioxidant activity, in the seeds and sprouts of mung beans, radish, broccoli and sunflower.

2. Materials and methods

2.1. Chemicals

Methanol, sodium carbonate, and potassium persulfate were purchased from POCh (Gliwice, Poland). Other reagents (aluminium chloride, sodium nitrate, sodium hydroxide, ascorbic acid and acetic acids, iron(III) chloride, hydrochloric acid, di-sodium EDTA, sodium acetate) were obtained from Chempur (Piekary Śląskie, Poland). Folin–Ciocalteau reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), TPTZ (2,4,6-tri(2-pyridyl-s-triazine)) were purchased from Sigma–Aldrich Chemie (Steinheim, Germany). All standards of phenolic acids and flavonoids were from Sigma–Aldrich Chemie or Fluka Chemie AG (Buchs, Switzerland).

2.2. Materials

Seeds of mung bean (Vigna radiata L. Wilczek), radish (Raphanus sativus L. var. ‘Flamboyant 2’), broccoli (Brassica oleracea L. var. italica ‘Ramoso Calabrese’) were purchased from Diet Food (Warsaw, Poland), and sunflower (Helianthus annuus L.) seeds from KHNO POLAN Ltd (Krakow, Poland).

2.2.1. Seed germination

Seeds were sterilised for 1 min by immersion in ethanol. Then the seeds were steeped in deionised water in ratio 1:10 (m/v) for 12 h. After pouring off the soaking water, the seeds were spread on sterile stackable trays (three for one species) and were washed twice a day using deionised water to avoid microbial growth. Sunflower seeds were initially peeled of their shells.
Germination of the seeds was carried out at room temperature in the range of 22 ± 1 °C (12/12 h day/night). Sprouted seeds were harvested after 5 days of growth. The seeds and their sprouts were lyophilised with an Alpha 1–4 freeze-dry system (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany), ground in a knife mill Grindomix GM 200 (Retsch, Düsseldorf, Germany) and stored in the dark until further analyses.

2.3. Procedure of extraction

Methanol extraction was carried out according to the method of Khattak, Zeb, Bibi, Khalil, and Khattak (2007). The ground seeds (5 g) and sprouts (3 g) were extracted for 20 min by shaking with 20 ml of methanol (99.8%) in a screw-capped tube. Extractions were carried out three times. The combined extracts were centrifuged at 10 000 rpm for 10 min and the organic solvents were removed at 35 ± 3 °C using a rotary vacuum evaporator RVO 200A (INGOS Laboratory Instruments Ltd., Prague, Czech Republic). The obtained residue was dissolved in 10 ml of methanol (99.8%) and stored in a freezer until analysis. The extraction procedure was carried out in triplicate.

2.3.1. Determination of total phenolic content

Total phenolic content of methanolic extracts was assayed as described by Meda, Lamien, Romito, Millogo, and Nacoulma (2005), using Folin–Ciocalteau reagent with final reaction measurements carried out at 760 nm. A 0.1-ml aliquot of methanolic extract was diluted with 0.4 ml of deionised water, then the obtained solution was mixed with 2.5 ml of 0.2 M Folin–Ciocalteau reagent and 2 ml of 7.5% (m/v) sodium carbonate solution. After 2 h of incubation, the absorbance was measured against a blank, using UV/Vis spectrophotometer V-530 (Jasco, Tokyo, Japan). Total phenolic content was expressed as mg of gallic acid equivalents (GAE) per g dry matter (d.m.) of seeds and sprouts.

2.3.2. Determination of total flavonoids content

Total flavonoid content in seeds and sprouts was analysed by a spectrophotometric method described by Boateng, Verghese, Walker, and Ogutu (2008). The method is based on the reaction between the flavonoids and aluminium chloride, forming a yellow complex. Four millilitres of deionised water and 0.3 ml of sodium nitrate solution (15 g/100 ml) were added to 1 ml of appropriately diluted methanol extract. After that 0.3 ml of aluminium chloride methanolic solution (10 g/100 ml) and 4 ml of sodium hydroxide solution with concentration (4 g/100 ml) were added to the resulting solution and then the whole sample was diluted with deionised water to a final volume of 10 ml. The mixture was stirred and left to stand for 15 min and finally absorbance was measured at 510 nm. The total flavonoids content in the extracts was compared to the standard curve for quercetin solutions and expressed as mg of quercetin equivalents (QE) per g d.m. of seeds and sprouts.

2.3.3. Determination of ABTS cation radical-scavenging activity

Determination of ABTS cation radical-scavenging activity was based on the reduction of the ABTS cation radical (dissolved in phosphate buffered saline (PBS)) by methanolic extracts from seeds and sprouts according to the method of Martinez-Villaluenga et al. (2010). ABTS cation radical was obtained in the reaction of 2 mM phospate-buffered stock solution of 2,2′-azino-bis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate. The mixture was left to stand for 24 h, until the reaction was completed and then ABTS solution was dilluted by PBS to obtain an absorbance of 0.800 ± 0.03 at λ = 734 nm. Fifty microlitres of appropriately diluted methanolic extract of seeds or sprouts was mixed with 6 ml of the ABTSradical dot+ solution and the absorbance of the resulting solution was measured after 15 min at 734 nm. Antioxidant activity was expressed as mg of Trolox equivalents per g d.m. of seeds and sprouts.

2.3.4. Determination of DPPH radical-scavenging activity

In order to determine DPPH radical-scavenging activity a method described by Moure et al. (2001) was used with minor modification. An aliquot of 3.9 ml of 0.1 mM DPPH radical in methanol was mixed with 0.1 ml of methanolic extract of the sample. After 60 min of incubation the absorbance of the sample was measured at 515 nm in a UV/Vis spectrophotometer (V-530; Jasco, Tokyo, Japan). The DPPH radical-scavenging activity in the extracts was expressed as mg of Trolox equivalents per g d.m. of seeds and sprouts.

2.3.5. Determination of ferric reducing antioxidant power (FRAP)

The ferric ion reducing activity of the methanol extracts was measured according to the method of Benzie and Strain (1996) with some modifications. At low pH, when a ferric-tripyridyltriazine (FeIII-TPTZ) complex is reduced to the ferrous (FeII) form, an intense blue colour develops with an absorption maximum at 593 nm. To 3.3 ml of acetate buffer (pH 3.6) consisting of 18.5 ml 0.2 M CH3COOH and 1.5 ml 0.2 M CH3COONa, 0.33 ml 20 mM FeCl3 and 0.33 ml 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM HCl were added. After 5 min of incubation at 37 °C, an aliquot of 0.33 ml of methanol extract was added to the mixture and the absorbance was measured at 593 nm after a further 15 min of incubation at 37 °C. Ferric reducing antioxidant power was expressed as mmol of Fe2+ per 100 g d.m. of seeds and sprouts.

2.3.6. Free phenolic acids and flavonoids

Free phenolic acids and flavonoids were analysed by high-performance liquid chromatography (HPLC; LaChrom, Merck-Hitachi, Tokyo, Japan) using UV detection, according to the method described by Socha et al. (2011) with minor modifications. The samples were separated on an ODS Hypersil column (250 × 4.6 μm × 5 μm, Thermo Fisher Scientific Inc., Waltham, MA) at a temperature of 30 °C. Phenolic acids, such as gallic, protocatechuic, syringic and vanillic were detected at 280 nm, and chlorogenic, ferulic, caffeic, p-coumaric and synapic acids at 320 nm. Flavonoids (quercetin, kaempferol, apigenin and luteolin) were monitored at 360 nm. Chromatographic separation was performed with gradient elution at a flow rate of 1 ml min−1 using two solvents: A – 2.5 g per 100 ml of acetic acid, and B – acetonitrile, as mobile phases. The chromatographic analysis was conducted as follows: for the first 10 min a linear gradient was applied, with mobile phase B increasing from 3% to 8%, followed by increase in phase B to 15%, 20%, 30% and 40% at 20, 30, 40 and 50 min, respectively. Finally the column was eluted isocratically for 10 min before the next injection. Before the chromatographic analyses, methanol extracts were filtered using 0.45 μm Millex-LCR syringe filters (PTFE), then purified using Hyper Sep C18 columns (500 mg, 6 ml; Polygen, Gliwice, Poland) and appropriately diluted. The qualitative measurement of the individual phenolic acids was based on comparison with standards from Sigma–Aldrich Chemie and Fluka Chemie AG. The calibration curves of the analysed phenolics were made in triplicate for each individual standard and were plotted separately for each standard at concentrations in the range of 0.02–0.2 mg/ml.

2.4. The hydrolytic procedure and extraction of bound phenolic compounds

The hydrolytic procedure of bound phenolic compounds from seeds and sprouts was carried out according the method of Ross, Beta, and Arntfield (2009). The ground sample (0.5 g) was mixed with 7 ml of methanol containing 10% acetic acid (85:15 v/v). The mixture was sonicated for 30 min and its volume was adjusted to 10 ml with deionised water. The obtained extract was mixed with 10 ml of deionised water and 5 ml of 10 M NaOH (containing 2% ascorbic acid and 13.4 mM EDTA). The mixture was flushed with argon and stirred for 16 h at ambient temperature. The pH of the solution was carefully adjusted to the value of 2 by dropwise addition of 6 M HCl and then the solution was saturated with NaCl.
Extraction of phenolic compounds was carried out as was previously described by Nardini and Ghiselli (2004). The liberated phenolic compounds were extracted three times with 25 ml of ethyl acetate. The mixture was combined and centrifuged for 10 min at 10 000 rpm (MPW-350; MPW Med. Instruments, Warsaw, Poland). The upper organic layer containing the phenolic compounds liberated during alkaline hydrolysis was collected from the bottom aqueous residue layer by pipetting. Ethyl acetate was evaporated to dryness using a rotary evaporator at 35 ± 3 °C. The residue was dissolved in 5 ml of methanol and stored at −18 °C in a test tube with stopper. Bound phenolic acids and flavonoids were analysed by HPLC according to the procedure described in Section 2.3.6.

2.5. Statistical analysis

The results were subjected to a one-way analysis of variance, and the least significant difference (LSD) using Fisher test at significance level 0.05 was calculated. The Pearson’s linear correlation coefficients between selected parameters were also calculated.

3. Results and discussion

3.1. Total phenolic content

Cereals and vegetables (including seed and sprouts) are a good source of phenolic compounds. According to literature data broccoli and radish are rich in anthocyanins, phenolic acids and flavonols (in particular, kaempferol and quercetin) (Koh, Wimalasiri, Chassy, & Mitchell, 2009). Broccoli sprouts are even richer in phenolic compounds than commercial broccoli florets (Vallejo, Tomás-Barberán, & García-Viguera, 2002). Legumes contain high concentration of isoflavones (Lin & Lai, 2006). Predominant phenolics in sunflower seeds are chlorogenic, quinic and caffeic acids (Weisz, Kammerer, & Carle, 2009). Germination resulted in significant changes in the phenolic composition, due mainly to activation of endogenous enzymes and the complex biochemical metabolism of seeds during this process (Dueñas et al., 2009).
Fig. 1 presents total phenolic content (expressed as mg GAE g−1) in analysed seeds and sprouts. Germination increased the total phenolic content of most seeds in order: mung bean (841%) > sunflower (232%) > radish (206%) > broccoli (68%). These results are in agreement with those published by Cevallos-Casals and Cisneros-Zevallos (2010), who reported that mung bean was the seed with the greatest increase in phenolics during germination compared to twelve other edible seed species. However, in our study, the increase of TP content in mung bean sprouts was lower (841%) than those observed by Cevallos-Casals and Cisneros-Zevallos (2010), who also reported that the phenolic contents decreased in the following order: mung bean > sunflower > broccoli > radish. These differences may result from the diversity among varieties, growing and storage conditions, as well as the extraction procedures (Gawlik-Dziki and Kowalczyk, 2007, Luthria and Pastor-Corrales, 2006, Naczk and Shahidi, 2004 and Ross et al., 2009).
Fig. 1.
Total phenolic content in seeds and sprouts, expressed as gallic acid equivalent (GAE) in mg per 1 g of plant dry mass.
Figure options
Paśko et al. (2009) also reported higher total phenolic content in sprouts compared to seeds, suggesting that synthesis of phenolic antioxidants during germination may occur. It is thought that seeds mainly act as a reservoir for the development of the sprouts (Pérez-Balibrea, Moreno, & García-Viguera, 2011).

3.2. Total flavonoid content

The total flavonoid content in seeds and sprouts determined as quercetin equivalents is presented in Fig. 2. The greatest quantity of flavonoids content was found in sunflower sprouts (45.6 mg/g d.m.), followed by broccoli (37.1 mg/g d.m.) and radish (34.8 mg/g d.m.) sprouts. The lowest amount of these compounds was determined in mung beans sprouts (13.7 mg/g d.m.). A similar trend was observed for the seeds, however those values were almost 50% lower than in sprouts.
Fig. 2.
Total flavonoids content in seeds and sprouts determined as quercetin equivalents (QE) in mg per 1 g of plant dry mass.
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The total flavonoids content determined in this study was in accordance with the results of Kim, Jeong, Gorinstein, and Chon (2012). These authors found that germination of mung bean increased (nearly three times) the flavonoid level, compared to the dry seeds, and this increment depended on the mung bean variety. These results agree with those reported by Dueñas et al. (2009), who observed twofold increase of flavones, dihydroflavonols and isoflavones in lupin sprouts after ten days of germination.

3.3. Antioxidant activity

An increase in phenolic compounds content along with the seeds germination may influence their free radical scavenging activity. The methanolic extracts of the seeds and sprouts studied were analysed in respect to their antioxidant activity against ABTS and DPPH radicals. The ferric ion reducing activity of the methanolic extracts (FRAP method) was also measured. The results are presented in Table 1.
Table 1. Antioxidant activity of mung bean, broccoli, radish and sunflower seeds and sprouts.
Sample
Antioxidant activity
ABTSradical dot+ [mg Trolox/g d.m.]DPPHradical dot [mg trolox/g d.m.]FRAP [mmol Fe2+/100 g d.m.]
SeedsMung bean0.86 ± 0.020.11 ± 0.000.13 ± 0.01
Radish10.52 ± 0.103.10 ± 0.057.50 ± 0.09
Broccoli8.86 ± 0.293.10 ± 0.077.10 ± 0.29
Sunflower7.75 ± 0.645.91 ± 0.424.69 ± 0.14
SproutsMung bean11.33 ± 0.341.41 ± 0.111.20 ± 0.01
Radish24.67 ± 0.066.07 ± 0.1410.49 ± 0.50
Broccoli12.33 ± 0.273.65 ± 0.318.64 ± 0.16
Sunflower18.38 ± 0.1611.47 ± 0.0511.05 ± 0.10
LSD0.050.520.350.43
Table options
Antioxidant activity of seeds was generally found to increase during germination. The sprouts of radish demonstrated the highest antioxidant activity, evaluated using the ABTS method, followed by sunflower sprouts. Mung bean sprouts exhibited the lowest AA, while broccoli sprouts demonstrated only slightly higher free radical scavenging activity. An increase in the antioxidant activity during the germination process was also observed in the reaction with DPPH free radical. However, in this case the sunflower sprouts exhibited the highest antioxidant capacity, followed by radish, broccoli and mung bean. It was also observed that the values of antioxidant activity increased almost twelve-fold for mung bean, twice for radish and sunflower, and by one-fifth in the case of broccoli sprouts, when compared to the seeds. Samotyja, Zdziebłowski, Szlachta, and Małecka (2007) have also observed that sunflower sprouts exhibited the highest AA against DPPH compared to radish, mung bean, wheat and lentil sprouts. The improvement of antioxidant activity during germination of broccoli and radish seeds up to the 4th day of germination was also reported by Martinez-Villaluenga et al. (2010). The increment of AA values during germination seems to be related to the rise in the content of antioxidant compounds, such as vitamins and polyphenols. However, these authors recorded a decrease in AA as germination proceeded.
There were high and significant linear correlations (α = 0.05) between total polyphenols content and antioxidant activity evaluated using ABTS and FRAP assays (r = 0.954 and 0.908, respectively), and these results suggest that phenolic compounds are good predictors of in vitro antioxidant activity ( Paśko et al., 2009). The weaker but still statistically significant (r = 0.654) correlation between total phenolic content and antioxidant activity measured in the reaction with DPPH radical suggests, that non-phenolic compounds, for example tocopherols or L-ascorbic acid, may be potential scavengers of DPPH radicals. Moure et al. (2001) reported that in beans among the compounds that have been reported to act as antioxidants are water soluble proteins, peptides and amino acids, as well as oligosaccharides from the hemicellulosic fractions. Lower, but also statistically significant correlations (α = 0.05) between total flavonoid content and AA determined using ABTS, DPPH and FRAP methods (r = 0.760, 0.868 and 0.846, respectively) were observed.
Influence of germination process on the ferric reducing antioxidant power (FRAP) of the analysed samples was significant. Higher antioxidant potential (FRAP) was found in sprouts. The germination process resulted in almost ten-fold increase of ferric reducing antioxidant power in mung bean and over twice for sunflower seeds. Broccoli and radish sprouts had only about 20% and 40% (respectively) greater reducing ability than their seeds. Paśko, Sajewicz, Gorinstein, and Zachwieja (2008) reported higher antioxidative activity (AA) in amaranthus and lower in quinoa sprouts (grown in daylight) compared to AA of sprouts. The AA of the amaranth sprouts ranged from 10.3 to 24.8 mmol Fe2+/100 g d.m., whereas AA of the quinoa ranged from 4.2 to 7.7 mmol Fe2+/100 g d.m. (depending on the time of growth and varieties). The AA of the sprouts was several times higher than that of seeds. An increase of in antioxidative activity after germination was also observed by Dueñas et al., 2009 and Martinez-Villaluenga et al., 2010, and Paśko et al. (2009). This process is attributed to the biochemical metabolism of seeds during germination (Dueñas et al., 2009). Higher AA of sprouts compared to seeds may result from differences in the content of polyphenols, anthocyanins and other compounds (Paśko et al., 2009). Samotyja et al. (2007) reported that sunflower sprouts had the highest antioxidative activity (estimated by the FRAP method), followed by radish sprouts. Mung bean sprouts had much less AA than sunflower and radish sprouts, and these data confirm results obtained in our study.

3.4. The profile of free phenolic acids and flavonoids

Phenolic acids occur in plants in different forms, such as aglycones (free phenolic acids), esters, glycosides, and/or bound complexes (Ross et al., 2009). In this study free phenolic acids extracted with methanol were determined (Table 2).
Table 2. Free phenolic acids and flavonoids content of seeds and sprouts extracts (mg/100 g d.m.) determined by HPLC.
Name of sample
Phenolic acids
TotalFlavonoids
Total
GallicProtocatechuicCaffeicp-CoumaricFerulicChlorogenicSinapicQuercetinKaempferolLuteolinApigenin
[mg/100 g d.m.]
[mg/100 g d.m.]
SeedsMung bean0.30n.d.0.03a0.020.22an.d.0.150.720.020.01n.d.n.d.0.03
Radish1.31a1.43a3.74b1.882.10n.d.010.46n.d.n.d.n.d.n.d.n.d.
Broccoli1.57a1.33a3.25b0.841.1712.02a0.56ab20.740.38an.d.n.d.n.d.0.38
Sunflower1.12a5.082.550.251.69n.d.1.5612.250.15a0.05n.d.0.290.49
SproutsMung bean4.55bn.d.0.25a0.142.641.081.299.950.24a0.180.10.190.71
Radish5.29b3.34b9.61c1.210.22an.d.0.65b20.327.090.10n.d.n.d.7.19
Broccoli16.103.84b6.960.700.39a11.49a0.46a39.940.29a0.091.610.782.77
Sunflower2.547.539.51c1.712.93n.d.3.2427.460.37a0.08n.d.n.d.0.45
Within columns, values with the same superscripts do not differ significantly at p < 0.05.
Table options
It was found that among free phenolic acids, caffeic, chlorogenic and gallic acids occurred in the largest amounts (both in seeds and sprouts). Among the studied samples mung bean seeds and sprouts exhibited the lowest total phenolic acids content and broccoli the highest. An increase in total phenolic acids content was observed with the germination process for all seeds. The total phenolic acids content ranged from 0.72 mg/100 g d.m. in mung bean seeds to 9.95 mg/100 g d.m. in mung bean sprouts (where gallic and ferulic acids were the predominant compounds). Luthria and Pastor-Corrales (2006) identified only three phenolic acids – p-coumaric, ferulic and sinapic – in fifteen edible beans; however, caffeic acid was identified in quantifiable amounts in only two varieties. According to these authors the total phenolic acids content among all 15 dry bean samples varied between 19.1 and 48.3 mg/100 g and was much higher than the values found in the seeds we studied. Higher content of phenolic acids (ca. 4–8 mg/100 g d.m. depending on the variety) in comparison to our results, was also found in three different lupin species studied by Siger et al. (2012). The authors reported that there was variability in the quantitative compositions of phenolic acids in seeds between the lupin species.
The sunflower seeds and sprouts investigated in our study had diverse phenolic acids profiles., with caffeic and protocatechuic acids being predominant in sunflower extracts. Germination increased the total phenolic acids over twice from 12.3 mg/100 g d.m. in seeds to 27.5 mg/100 g d.m. in sprouts. Cevallos-Casals and Cisneros-Zevallos (2010) reported that sunflower sprouts seem to have the one of the highest antioxidant activities among fruits and vegetables. The major phenolic compounds identified by these authors in sunflower seeds were chlorogenic acid (and its derivatives) and caffeic acid. They found a high phenolic content in radish, broccoli, mustard and fava seeds. Broccoli and mustard sprouts after 7-days of germination had the highest phenolic compounds content among the samples.
The predominant acid determined in broccoli seeds was chlorogenic, while gallic, chlorogenic and caffeic acids were identified in sprouts. These results are not in accordance with the data reported by Pérez-Balibrea et al. (2011), where the main free phenolic acids found in broccoli seeds and sprouts were ferulic and sinapic acids. The concentration of these compounds was much higher than in our study and ranged from 18.9 to 35.2 mg/100 g of fresh weight, depending on the time of growth and broccoli variety. Gawlik-Dziki et al. (2012) reported that ethanolic extracts of broccoli sprouts revealed considerable amounts of hydroxycinnamic acid derivatives, such as sinapinic and ferulic acid (17.9 and 4.70 μg/g f.w., respectively) and chlorogenic acid (13.3 μg/g f.w.). In that study significant amounts of benzoic acid and its derivatives, including salicylic and gallic acids were also found in broccoli sprouts.
The differences between phenolic contents reported in various studies could result from multiple factors, such as methodology (procedure of extraction, different susceptibilities to degradation, type of chromatography and quantification), plant species, growth and storage environments (Naczk and Shahidi, 2004, Pérez-Balibrea et al., 2011 and Ross et al., 2009). However, the results obtained in our study show that mung bean seeds and sprouts were the poorest in phenolic compounds and exhibited the lowest AA among the samples studied and these results are in accordance with the literature data (Cevallos-Casals, & Cisneros-Zevallos, 2010). The increases in phenolic content and antioxidant capacity show potentially important roles of phenolics during germination, as well as potential enhancement of nutraceutical value of seeds by the germination process (Cevallos-Casals, & Cisneros-Zevallos, 2010).
The following free flavonoids were identified in the studied seeds and sprouts: quercetin, kaempferol, luteolin and apigenin. The amounts of the flavonoids presented in Table 2 were inconsiderable both for seeds and for sprouts. Radish sprouts were the exception, because they contained 7.19 mg of flavonoids per 100 g of dry mass. There were no free flavonoids detected in radish seeds; however, considerable amounts of flavonoids were observed after alkaline hydrolysis (Table 3). The predominant flavonoid in radish sprouts was quercetin. Luteolin was identified in broccoli and mung bean sprouts, while apigenin was detected in sunflower seeds, mung bean and broccoli sprouts. Karamać, Kosińska, Estrella, Hernández, and Dueñas (2012) also identified several flavonoids in six different fractions of sunflower seeds. These compounds were classified as quercetin derivatives, diglycoside, rutinoside, glucoronide and flavanone. The sum of these compounds ranged from 0 to 9.84 mg/g depending on the fraction. Paśko et al. (2008) identified no flavonoids in Amaranthus cruentus v. Aztek seeds, but found vitexin, and isovitexin in the seeds of amaranth v. Rava (total amount of 67.6 mg/100 g d.m.), and orientin, vitexin, rutin, morin and traces of hesperidin and neohesperidin in quinoa seeds (total amount of 224 mg/100 g d.m.). In the case of amaranthus v. Aztek and quinoa the total amounts of flavonoids were higher in sprouts grown in daylight than in the seeds. These amounts were much higher than the values obtained in our study for seeds and sprouts of mung bean, radish, broccoli and sunflower. The main phenolic compounds found in extract of green lentil seeds by Troszynska et al. (2011) were catechin, quercetin and kaempferol derivatives. The sprouts were found to be rich in acylated flavonol glycosides, with the maximum content of polyphenols determined after seven days of germination. The results obtained by the abovementioned authors indicate that germination is a process which modifies both qualitative and quantitative polyphenolic composition of seeds over time, resulting in a significant increase in flavonoids content.
Table 3. Bound phenolic acids and flavonoids content of seeds and sprouts extracts (mg/100 g d.m.) determined by HPLC.
Name of sample
Phenolic acids
TotalFlavonoids
Total
GallicProtocatechuicVanillicCaffeicp-CoumaricFerulicChlorogenicSinapicQuercetinKaempferolLuteolinApigenin
[mg/100 g d.m.]
[mg/100 g d.m.]
SeedsMung bean0.54an.d.n.d.1.29a1.06an.d.n.d.22.46a25.351.09abcn.d.0.36an.d.1.45
Radish3.133.9811.74.50a6.09a35.64n.d.994c10591.36ab0.503.54bn.d.5.40
Broccoli0.36a7.61n.d.2.03a0.69a1.581.47566b5790.99ac0.42a1.237.5110.15
Sunflower1.30b4.82n.d.7491.27an.d.n.d.1.55a7580.43cn.d.n.d.n.d.0.43
sproutsMung bean1.29b5.41n.d.79.567.7113.50n.d.15.2a1830.54bc0.110.35a0.44a1.44
Radish3.85n.d.n.d.3.78a3.11a12.32n.d.948c9711.38a0.37a3.39bn.d.5.14
Broccoli4.127.782.424.12a2.04a7.66n.d.548b5768.940.645.08n.d.14.66
Sunflower2.26n.d.n.d.10423.08a5.10n.d.3.57a10560.76abcn.d.0.63a0.23a1.62
Within columns, values with the same superscripts do not differ significantly at p < 0.05.
Table options
The quantitative determination of the sum of phenolic acids and flavonoids evaluated chromatographically did not compare directly with the total phenolic content (Fig. 1). However, a significant correlation was observed (r = 0.729). Similar results were reported by Siger et al. (2012) for lupin seeds. The difference could result from a lack of selectivity of the colorimetric Folin–Ciocalteu method, as well as from the fact that the assay used for the total phenol content determination can interfere with other compounds (e.g. nitric compounds, saccharides) ( Siger et al., 2012). Statistically significant correlation (α = 0.05) between the sum of phenolic acids and flavonoids evaluated chromatographically vs. antioxidant activity evaluated by the FRAP method ( Table 1) was also observed in this study (r = 0.773).

3.5. The profile of bound phenolic acids and flavonoids

There is information in the literature concerning the extraction and analysis of polyphenols from plant materials, including seeds and sprouts. However, most of it concerns aglycones (free phenolic acids), esters and glycosides (Dueñas et al., 2009, Lin and Lai, 2006, Martinez-Villaluenga et al., 2010, Troszyńska et al., 2011 and Weisz et al., 2009). There is scarce information concerning complexes of phenolic acids with cell wall polymers. The bound phenolics are not extractable by organic solvents, since the ester and glycosidic bonds between phenolic acids and cell wall polymers are not hydrolysed by these solvents. Bound phenolic acids are typically liberated from such complexes using alkaline hydrolysis, acid hydrolysis or both (Ross et al., 2009). In this work alkaline hydrolysis (10 M NaOH) with ascorbic and ethylenediaminetetraacetic (EDTA) acids protection was performed. Ross et al. (2009) recommended this method, as it allows the majority of phenolic acids to be detected.
Table 3 shows the bound phenolic acids content of seeds and sprouts determined by HPLC. Insignificant amounts of free phenolic acids were extracted in the free phenolic acid fraction (Table 2). However alkaline hydrolysis of the seeds and sprouts extracts, provided the majority of the phenolic acids. The amount of total bound phenolic acids increased after germination only for mung bean and sunflower (from 25.4 to 183 and from 758 to 1056 mg/100 g d.m., respectively), while broccoli remained almost unchanged and for radish a small decrease in total bound phenolic acids was observed. The seeds of mung bean contained mainly sinapic acid, although slight amounts of gallic, caffeic and p-coumaric acids were also detected. After germination mung bean showed much larger amounts of these acids. Moreover, ferulic and protocatechuic acids were also identified. Caffeic and p-coumaric were the predominant acids in mung bean sprouts. Broccoli and radish seeds contained mainly sinapic acid (566 and 994 mg/100 g d.m., respectively) and values did not increase after germination. The other phenolic acids identified in broccoli seeds and sprouts were gallic, protocatechuic, vanillic (the last one not detected in seeds), caffeic, p-coumaric, ferulic and chlorogenic (not detected in sprouts). Radish sprouts contained lower concentration of caffeic, p-coumaric, and ferulic acids than seeds, whereas protocatechuic and vanillic acids identified in seeds were not found in sprouts.
The flavonoids contents after alkaline hydrolysis of seeds and sprouts are presented in Table 3. The following flavonoids were identified: quercetin, kaempferol, luteolin and apigenin. Hydrolysis improved extraction of phenolic compounds from the materials. Comparing the results presented in Table 2 and Table 3, a significant increase of total flavonoids content after hydrolysis was observed. Germination increased the bound flavonoids level only in the case of broccoli and sunflower. The main flavonoid identified in the radish seeds and sprouts was luteolin. Apigenin was detected in broccoli seeds, while quercetin and luteolin were present in broccoli sprouts. The largest amount of bound flavonoids content was found in broccoli seeds and sprouts, while the smallest amount was in mung bean seeds, and sunflower seeds and sprouts. Statistically significant linear correlation (α = 0.05) between the sum of bound phenolic acids and flavonoids evaluated chromatographically vs. antioxidant activity evaluated by DPPH and FRAP methods ( Table 1) was observed in this study (r = 0.754 and 0.874, respectively).

4. Conclusions

The comparison of seeds and sprouts from different botanical sources is very important, due to the fact that they vary in their polyphenolic composition and antioxidant activity. Mung bean seeds are the cheapest and germinate the easiest, when compared to radish, broccoli and sunflower seeds. However, both seeds and sprouts of this plant exhibit the lowest antioxidant activity and phenolic contents, being lower than those of radish, broccoli and sunflower seeds. Germination significantly increases the levels of phenolic acids and flavonoids, as well as their antioxidant activity. Therefore, germinated edible seeds are a very valuable source of natural antioxidants. Lyophilised sprouts could be used as ingredients in functional foods.

Acknowledgement

The scientific research was realized within a framework of grant nr BM-4726/KAiOJŻ/2012 financed from budget of University of Agriculture in Krakow, Poland. The part of results were presented at the 2nd International Professional Conference On “Trends and Challenges in Food Technology, Nutrition, Hospitality and Tourism”, November 16 And 17, 2012, Ljubljana, Slovenia.

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