Scientia Horticulturae 140 (2012) 87–95

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Impact of germination time and type of illumination on the antioxidant

compounds and antioxidant capacity of Lens culinaris sprouts
Michał Świeca ∗ , Urszula Gawlik-Dziki, Dariusz Kowalczyk, Urszula Złotek
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Lentil sprouts have the potential to be an effective dietary source of polyphenolics. The effects of differ-
Received 7 October 2011 ent illumination conditions of germination on the content of polyphenolics and antioxidant capacities of
Received in revised form 29 March 2012 lentils sprouts have been studied. Obtained results indicated that both germination and illumination
Accepted 2 April 2012
conditions modified the polyphenolic profiles and biological activity. Studies have shown that phe-
nolic phytochemicals can be stimulated by cultivation under continuous light; a significant increase
Keywords:
in p-hydroxybenzoic, benzoic, caffeic acids content on days 3 and 4 after germination was observed.
Antioxidant activity
Antioxidant activity of sprouts was correlated with phenolics content and closely depended on the cul-
Cultivation condition
Elicitation
tivation conditions. To achieve lentil sprouts with enhanced nutraceutical value (antioxidant potential),
Lentil germination in the presence of continuous light can be suggested.
Sprouts © 2012 Elsevier B.V. All rights reserved.

1. Introduction at the molecular level and modification of metabolic pathways

with elicitors seem to be an alternative for genetic modification
In recent years, food legumes have attracted a great deal (GMO) not acceptable to many consumers (Shetty, 2004). It is
of attention due to their functional components and health- well known that the content of phenolics in legumes sprouts is
promoting effects in relation to the prevention of chronic diseases generally affected by sprouting environmental and genetic fac-
which include cardiovascular diseases, obesity, diabetes, inflam- tors such as cultivar, illumination and temperature (Lin and Lai,
mation and cancer (Losso, 2003; Caccialupi et al., 2010). Etiology 2006). Phenolics are primarily produced through the pentose phos-
of these pathological disorders and diseases is inter alia bound phate pathway, shikimate and phenylpropanoid pathway. Recent
with oxidative damage, caused by reactive oxygen species (ROS) evidence suggests that phenolics play an important role in the reg-
(Ferrari and Torres, 2003; Møller and Loft, 2006). ROS such as O2 •− , ulation of plant metabolism, e.g., acting as signaling molecules,
H2 O2 , and • OH are incessantly generated inside the human body as regulators of auxin transport (Shetty, 2004).
a consequence of the exposure to multitude of exogenous chemi- So far, little has been reported about the effect of different
cals in our ambient environment and/or a number of endogenous illumination conditions on chemical composition, and antioxidant
metabolic processes involving redox enzymes and bioenergetics activities of legume sprouts. Stimulation of phenolic-antioxidant
electron transfer (Zhao et al., 2005). production has been observed in faba bean after UV-light exposure
Many studies have revealed that the phenolic contents of (Shetty et al., 2002; Randhir and Shetty, 2003), chickpea followed by
sprouts can be correlated with their antioxidant activities and gamma irradiations and colored illumination treatment (Khattak
nutraceutical properties (Scalbert and Williamson, 2000; Fang et al., 2007).
et al., 2002; Scalbert et al., 2005; Shivashankara and Acharya, 2010; The objectives of this study were to determine the influence of
Gawlik-Dziki and Świeca, 2011). Thus, in the last few years many illumination on the polyphenolic contents and their antioxidative
trails have been undertaken to modify the chemical composition of abilities at different germination stages of lentil. The information
functional foods including legumes sprouts. Metabolic engineering provided in this study is valuable for the selection of lentil germi-
nating conditions to develop functional foods.

Abbreviations: ABTS, radical scavenging ability; CHP, chelating power; CTC, 2. Materials and methods
condensed tannin content; D, darkness; DL, 12-h illumination and 12-h darkness
photoperiod; L, constant illumination; LPI, inhibition of linoleic acid peroxidation;
2.1. Materials
PAC, phenolic acids content; RP, reducing power; TFC, flavonoids content; TPC, total
phenolics content.
∗ Corresponding author. Tel.: +48 81 4623327; fax: +48 81 4623324. Lentil seeds var. Tina were purchased from the PNOS S.A. in
E-mail address: michal.swieca@up.lublin.pl (M. Świeca). Ozarów Mazowiecki, Poland. Seeds were sterilized in 1% (v/v)

0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scienta.2012.04.005
88 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95

sodium hypochloride for 10 min, then drained and washed with nitrite dissolved in 100 ml of distilled water) and 1 ml 1 mol L−1
distilled water until they reached neutral pH. They were placed NaOH and distilled water was added to a final volume of 10 ml.
in distilled water and soaked for 6 h at 25 ◦ C. Seeds were germi- Absorbance was measured at 490 nm. The total phenolic acid con-
nated for 8 days in a growth chamber on Petri dishes ( 125 mm) tent was expressed as caffeic acid equivalent (CAE) in ␮g per g of
lined with absorbent paper under different light conditions: dark- fresh mass (FM).
ness (D), 12-h light and 12-h dark photoperiod (DL) and constant
light (L). Sprout samples were collected at different time points (1, 2.4.5. HPLC analysis
2, 3, 4, 6 and 8 days). For each day and growth condition, sprouts Phenolics were analyzed according to the method described by
were gently collected, rapidly frozen and stored in polyethylene Gawlik-Dziki and Świeca (2007). Samples were analyzed with a
bags at −20 ◦ C for the meal preparation. For each treatment, three Varian ProStar HPLC System separation module (Varian, Palo Alto,
replicates were taken for analysis. CA) equipped with Varian ChromSpher C18 reverse phase column
(25 mm × 4.6 mm) column and Pro star 325 UV-Vis detector. The
2.2. Dry weight to fresh weight correlation column thermostat was set at 40 ◦ C. The mobile phase consisted
of 4.5% acetic acid (solvent A) and 50% acetonitrile (solvent B) and
Lentil sprouts samples from various stages of germination were flow rate 0.8 ml min−1 . At the end of the gradient, the column was
measured for both their dry and fresh weight. Sprouts were freeze- washed with 50% acetonitrile and equilibrated to the initial condi-
dried. The correlation of dry weight to wet weight was determined tion for 10 min. Quantitative determinations were carried out with
as an amount of fresh mass obtained from 1 g of dormant seeds the external standard calculation, using calibration curves of the
during germination. standards. The gradient elution was used as follows: 0 min 92% A,
30 min 70% A, 45 min 60%, 80 min 61% A, 82 min 0% A, 85 min 0%
2.3. Extract preparation A, 86 min 92% A, and 90 min 92% A. Detection was carried out at
270 nm and 370 nm. Comparing their retention times with those
Sprouts were dried in a forced-air oven at 50 ◦ C for 12 h to a of the standard compounds identified phenolic compounds in a
moisture content <10% (wb). Dried sprouts were stored at 4 ◦ C until sample. Phenolics were expressed in mg per g of fresh mass (FM).
ground to flour (Ghavidel and Prakash, 2007). Extraction proce-
dure was conducted according to Xu and Chang (2007). Briefly,
2.5. Antioxidant activities
lentil flours (0.2 g in triplicate) were extracted three times with
4 ml of acetone/water/hydrochloric acid (70:29:1, v/v/v). After cen-
2.5.1. Antiradical activity (ABTS)
trifugation (10 min, 8000 rpm (6800 rcf)) fractions were collected,
The experiments were carried out using an improved ABTS
combined and used for further analysis.
decolorization assay (Re et al., 1999). ABTS+• was generated by
oxidation of ABTS with potassium persulfate.
2.4. Phenolics determination
The ABTS radical cation (ABTS+• ) was produced by reacting
7 mM stock solution of ABTS with 2.45 mM potassium persulfate
2.4.1. Determination of total phenolic compounds (TPC)
(final concentration) and allowing the mixture to stand in the dark
The amount of total phenolics was determined using Folin-
for at least 6 h at room temperature prior to use. The ABTS+• solu-
Ciocalteau reagent (Singleton et al., 1974). To 0.5 ml of the sample,
tion was diluted to an absorbance of 0.7 ± 0.05 at 734 nm (Lambda
0.5 ml H2 O, 2 ml Folin-Ciocalteau reagent (1:5 H2 O) were added,
40 UV-Vis spectrophotometer, Perkin Elmer). The affinity of test
and after 3 min, 10 ml of 10% Na2 CO3 and the contents were
material to quench ABTS free radical was evaluated according to
mixed and allowed to stand for 30 min. Absorption at 725 nm was
the following equation:
measured in a UV–vis spectrophotometer. The amount of total phe-
A − A 
nolics was calculated as a gallic acid equivalent (GAE) in mg per g C A
scavenging (%) = × 100
of fresh mass (FM). AC

where AC is the absorbance of control and AA is the absorbance of

2.4.2. Determination of flavonoids content (TFC)
sample.
Total flavonoid content was determined according to the
Free radical scavenging ability was expresses as IC50 in mg fresh
method described by Lamaison and Carnet (1990). One milliliter
mass per ml.
of extract was mixed with 1 ml of 2% AlCl3 × 6H2 O solution (in
methanol) and incubated at room temperature for 10 min. There-
after, absorbance at 430 nm was measured. Total flavonoid content 2.5.2. Reducing power (RP)
was calculated as a quercetin equivalent (QE) in mg per g of fresh Reducing power was determined by the method of Pulido et al.
mass (FM). (2000). Analyzed sample (2.5 ml) was mixed with phosphate buffer
(2.5 ml, 200 mM, pH 6.6) and potassium ferricyanide K3 [Fe(CN6 )]
2.4.3. Determination of condensed tannins content (CTC) (2.5 ml, 1%). The mixture was incubated at 50 ◦ C for 20 min. Reac-
Condensed tannin content was determined according to the tions were stopped with 0.5 ml 10% TCA and centrifuging for 10 min
method described by Sun et al. (1998). To 0.2 ml of extract, 1 ml of at 650 rpm. The upper layer of solution (2.5 ml) was mixed with
freshly prepared vanillin reagent 1% (w/v) vanillin in glacial acetic distilled water (2.5 ml) and 0.5 ml of 0.1% FeCl3 and the absorbance
acid:HCl solution (92:8) was added. After incubation for 20 min at was measured at 700 nm. Increased absorbance of the reaction mix-
30 ◦ C, the absorbance was measured at 510 nm. Condensed tannin ture indicated increase in reducing power. Reducing power was
content was calculated as a (+)-catechin equivalent (CE) in mg per expressed as quercetin equivalent (Q) in ␮g per mg of fresh mass
g of fresh mass (FM). (FM).

2.4.4. Determination of phenolics acids content (PAC) 2.5.3. Metal chelating activity (CHP)
Total phenolic acid estimation was carried out according to Chelating power was determined by the method of Guo et al.
the Arnov method (Szaufer-Hajdrych, 2004). One milliliter of sam- (2001). The extract samples (5 ml) was added to 0.1 ml of 2 mM
ple was mixed with 5 ml of distilled water, 1 ml 0.5 mol L−1 HCl, FeCl2 solution and 0.2 ml 5 mM ferrozine and the mixture was
1 ml of Arnov reagent (10 g sodium molybdate and 10 g sodium shaken vigorously and left standing at room temperature for
 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95 89

Fig. 1. Dry weight–fresh weight relationship in lentil sprouts cultivated under different illumination conditions (D, DL, L) Abbreviations: D, darkness; DL, 12-h illumination
and 12-h darkness photoperiod; L, constant illumination. Means followed by different small letters are significantly different at ˛ < 0.05.

10 min. Then, absorbance of the solution was measured spec- Hydroxyperoxide formation was assayed according to a ferric thio-
trophotometrically at 562 nm. The percentage of inhibition of cyanate method with mixing in order of 0.02 M FeCl2 (0.1 ml) and
ferrozine–Fe2+ complex formation was calculated according to the 30% ammonium thiocyanate (0.1 ml). Absorbance at 480 nm (As)
formula: was measured with spectrophotometer (Lambda 40, Perkin-Elmer)
 Ap
 for 5 min. The absorbance of blank (Ao ) was obtained without
% inhibition = 1 − × 100, the addition of hemoglobin to the above reaction mixture; the
Ac
absorbance of control (A100 ) was obtained with no sample addi-
where Ac is the absorbance of control and Ap is the absorbance of tion to the above mixture. Thus, the antioxidative activity of the
sample. sample was calculated as:
Chelating power was expressed as EDTA equivalent (EDTA) in
␮g per mg of fresh mass (FM). 1 − (As − A0 )
LPI (%) = × 100
A100 − A0
2.5.4. Inhibition of linoleic acid peroxidation (LPI)
The antioxidant activity was determined as the degree of inhi- Inhibition of linoleic acid peroxidation was expressed as
bition of the hemoglobin-catalyzed peroxidation of linoleic acid quercetin equivalent (Q) in ␮g per mg of fresh mass (FM).
according to Groupy et al. (2007). Ten microliters of sample was
mixed with 0.37 ml 5 mM phosphate buffer (pH 7) containing 2.6. Statistical analysis
0.05% Tween 20 and 4 mM linoleic acid and then equilibrated at
37 ◦ C for 3 min. The peroxidation of linoleic acid in the above- All experimental results were mean ± S.D. of three parallel
mentioned reaction mixture was initiated by adding 20 ␮l 0.035% measurements. Two factorial analysis of variance (ANOVA, Tukey
hemoglobin (in water) followed by incubation in a shaking bath at test) was used to compare groups within illumination condi-
37 ◦ C for 10 min and stopped by adding 5 ml 0.6% HCl (in ethanol). tion and time of germination. ˛ values < 0.05 were regarded as a

Table 1
Phenolic composition (TPC, TFC, PAC, CTC) of lentil sprouts cultivated under different illumination conditions (D, DL, L).

Phenolics Light Raw Soaked Time of germination (days)

conditions

1 2 3 4 6 8

D 8.01 ± 0.06 fg 7.97 ± 0.32 fg 7.11 ± 0.09 def 6.33 ± 0.59 cd 4.87 ± 0.13 ab 4.40 ± 0.03 ab
TPC mg g−1 FM DL 18.98 ± 0.39 i 9.67 ± 0.82 h 8.73 ± 0.58 g 7.30 ± 0.13 ef 6.96 ± 0.23 cde 6.68 ± 0.30 cde 5.07 ± 0.41 b 4.67 ± 0.15 ab
L 8.65 ± 0.41 g 8.59 ± 0.53 g 7.35 ± 0.27 ef 7.18 ± 0.35 def 7.17 ± 0.27 def 6.15 ± 0.26 c

D 0.72 ± 0.02 fgh 0.73 ± 0.02 fgh 0.67 ± 0.01 efg 0.54 ± 0.00 cd 0.42 ± 0.01 ab 0.38 ± 0.02 a
TFC mg g−1 FM DL 1.47 ± 0.07 k 0.87 ± 0.09 i 0.99 ± 0.02 j 0.99 ± 0.09 j 0.80 ± 0.02 hi 0.75 ± 0.02 gh 0.51 ± 0.05 bc 0.54 ± 0.01 cd
L 0.79 ± 0.06 hi 0.82 ± 0.02 hi 0.72 ± 0.03 fgh 0.67 ± 0.04 efg 0.63 ± 0.03 def 0.59 ± 0.06 cde

D 3.21 ± 0.08 h 2.42 ± 0.13 g 2.02 ± 0.08 f 1.57 ± 0.12 e 1.09 ± 0.00 bcd 0.91 ± 0.00 abc
CTC mg g−1 FM DL 10.64 ± 0.28 j 4.22 ± 0.27 i 2.01 ± 0.24 f 1.64 ± 0.01 e 1.50 ± 0.09 e 1.35 ± 0.11 de 0.74 ± 0.07 ab 0.60 ± 0.02 a
L 2.22 ± 0.01 fg 2.00 ± 0.12 f 1.54 ± 0.04 e 1.31 ± 0.15 de 1.38 ± 0.04 de 1.13 ± 0.05 cd

D 6.11 ± 0.53 cde 5.36 ± 0.32 c 4.46 ± 0.09 b 4.35 ± 0.27 b 3.25 ± 0.08 a 3.41 ± 0.23 a
PAC ␮g g−1 FM DL 13.98 ± 0.52 l 8.79 ± 0.27 i 11.20 ± 0.63 k 9.83 ± 0.70 j 8.26 ± 0.39 hi 7.44 ± 0.16 gh 6.89 ± 0.22 efg 6.97 ± 0.45 efg
L 7.24 ± 0.73 fg 7.40 ± 0.15 fgh 6.54 ± 0.08 def 5.91 ± 0.53 cd 5.87 ± 0.37 cd 5.40 ± 0.33 c

Abbreviations: D, darkness; DL, 12-h illumination and 12-h darkness photoperiod; L, constant illumination; CTC, condensed tannin content; PAC, phenolic acids content; TFC,
flavonoids content; TPC, total phenolics content.
Means followed by different small letters are significantly different at ˛ < 0.05.
 90
Table 2
Phenolic acids composition of lentil sprouts cultivated under different illumination conditions (D, DL, L).

Compounds (␮g g−1 FM) Light conditions Raw Soaked Time of germination (days)

1 2 3 4 6 8

D 3.01 ± 0.21 efg 1.77 ± 0.04 cd 0.73 ± 0.03 ab 0.69 ± 0.03 ab 1.00 ± 0.01 abc 0.48 ± 0.02 a
Gallic acid DL 8.25 ± 1.23 j 3.91 ± 0.34 gh 3.12 ± 0.09 fg 2.37 ± 0.32 def 1.29 ± 0.11 abc 1.02 ± 0.09 abc 1.61 ± 0.10 bcd 1.54 ± 0.21 bcd
L 3.32 ± 0.43 fgh 4.19 ± 0.11 h 1.55 ± 0.09 bcd 1.70 ± 0.15 bcd 2.00 ± 0.21 cde 1.11 ± 0.08 abc

D 7.87 ± 0.76 defg 6.34 ± 0.87 bcd 6.35 ± 0.12 bcd 5.66 ± 0.87 abc 5.49 ± 0.09 abc 6.83 ± 0.32 bcdef
p-Hydroxybenzoic acid DL 13.46 ± 1.78 h 9.50 ± 1.23 g 8.88 ± 1.02 fg 3.59 ± 0.42 a 6.74 ± 0.45 bcde 6.68 ± 0.21 bcde 6.37 ± 0.29 bcd 4.77 ± 0.11 ab
L 7.17 ± 1.32 cdef 5.47 ± 0.75 abc 6.96 ± 0.29 cdef 8.49 ± 0.59 efg 8.07 ± 0.33 defg 12.76 ± 0.99 h

D 1.06 ± 0.09 a 1.22 ± 0.11 abc 1.66 ± 0.19 abcd 1.25 ± 0.03 abc 4.34 ± 0.55 f 8.64 ± 0.98 h

M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95

Caffeic acid DL 5.55 ± 0.34 g 2.16 ± 0.07 de 2.02 ± 0.12 bcde 1.03 ± 0.02 a 1.16 ± 0.01 ab 2.88 ± 0.43 e 3.82 ± 0.23 f 10.36 ± 1.04 i
L 1.15 ± 0.07 a 2.29 ± 0.25 de 2.46 ± 0.19 de 2.07 ± 0.11 cde 6.05 ± 0.07 bB 17.87 ± 0.77 j

D 8.70 ± 0.22 d 6.31 ± 0.22 bcd 2.28 ± 0.11 a 17.04 ± 2.91 ef 31.19 ± 2.31 j 15.59 ± 1.22 e
Benzoic acid DL 24.27 ± 3.78 gh 4.49 ± 0.22 abc 2.79 ± 0.07 ab 3.79 ± 0.19 abc 3.20 ± 0.47 abc 28.75 ± 2.34 ij 20.48 ± 1.98 fg 25.17 ± 1.33 hi
L 8.49 ± 0.76 d 6.81 ± 0.71 cd 2.42 ± 0.32 ab 20.31 ± 1.22 f 38.85 ± 2.66 k 19.48 ± 0.99 ef

D 4.83 ± 0.21 gh 6.88 ± 0.22 ij 7.95 ± 0.71 j 4.50 ± 0.05 fgh 2.81 ± 0.11 cdef 0.50 ± 0.03 ab
Syringic acid DL 11.96 ± 0.06 k 26.74 ± 2.12 l 3.62 ± 0.07 defg 2.56 ± 0.31 cde 2.08 ± 0.03 bcd 1.17 ± 0.22 abc 1.69 ± 0.03 abc 4.01 ± 0.08 efg
L 3.54 ± 0.18 defg 6.07 ± 0.78 hi 3.62 ± 0.41 defg 0.68 ± 0.46 ab 0.02 ± 0.00 a 0.47 ± 0.04 ab

D 9.37 ± 1.01 bc 8.12 ± 0.56 b 9.29 ± 0.33 bc 10.24 ± 1.39 bcd 12.38 ± 2.45 de 11.57 ± 0.04 cd
Salicylic acid DL 1.10 ± 0.07 a 2.03 ± 0.41 a 7.82 ± 0.73 aA 8.19 ± 0.34 b 14.52 ± 1.34 e 23.44 ± 1.45 gh 21.57 ± 1.76 g 18.26 ± 2.78 f
L 8.42 ± 1.22 b 8.88 ± 0.99 bc 18.45 ± 1.02 f 22.61 ± 0.32 gh 32.87 ± 3.76 i 24.72 ± 0.66 h

D 4.39 ± 0.02 b 4.46 ± 0.33 b 5.68 ± 0.56 bc 2.50 ± 0.19 a 4.50 ± 0.11 b 3.56 ± 0.03 a
Sinapic acid DL 43.95 ± 6.11 d 9.68 ± 0.11 c 4.73 ± 0.32 ab 5.55 ± 0.76 bc 5.93 ± 0.11 bc 4.63 ± 0.34 b 4.58 ± 0.56 b 3.61 ± 0.10 a
L 2.45 ± 0.11 a 6.26 ± 0.32 bc 6.19 ± 0.89 bc 4.90 ± 0.09 b 4.33 ± 0.42 b 3.28 ± 0.21 a

D 20.65 ± 1.56 ij 17.62 ± 1.89 fgh 14.37 ± 1.22 efg 6.04 ± 0.77 abc 4.58 ± 0.33 abc 3.05 ± 0.05 a
Chlorogenic acid DL 44.46 ± 7.56 k 32.50 ± 1.43 j 25.77 ± 3.67 i 22.02 ± 1.67 hi 20.22 ± 1.45 ghi 15.74 ± 2.33 efgh 10.00 ± 0.72 bcde 6.86 ± 0.82 abc
L 12.96 ± 2.89 def 14.82 ± 0.77 efg 13.06 ± 0.99 def 14.09 ± 1.09 efg 3.99 ± 0.19 ab 10.90 ± 1.76 cde

D 24.19 ± 2.65 ef 22.22 ± 3.89 de 28.77 ± 3.42 ghi 24.45 ± 1.79 efg 28.23 ± 4.19 fgh 33.08 ± 4.00 i
Ferulic acid DL 18.95 ± 1.02 cd 28.55 ± 0.69 fgh 16.10 ± 2.01 bc 16.23 ± 0.22 bc 10.83 ± 0.99 a 20.24 ± 2.03 cde 31.08 ± 2.11 hi 44.35 ± 1.93 j
L 11.19 ± 1.76 a 15.80 ± 0.78 bc 10.31 ± 1.22 a 8.60 ± 0.45 a 10.36 ± 0.12 a 11.87 ± 2.01 ab

D 3.38 ± 0.29 def 4.95 ± 0.15 ij 5.09 ± 0.08 j 2.49 ± 0.19 bc 2.65 ± 0.11 bc 2.50 ± 0.19 bc
o-Coumaric acid DL 0.78 ± 0.02 a 6.04 ± 0.05 k 5.45 ± 0.41 jk 2.63 ± 0.38 bc 3.78 ± 0.70 efg 5.22 ± 0.34 j 4.01 ± 0.01 fgh 3.03 ± 0.34 cde
L 2.78 ± 0.33 cd 4.32 ± 0.22 ghi 9.14 ± 0.72 i 6.72 ± 0.63 l 4.41 ± 0.04 hi 2.02 ± 0.11 b

D 24.19 ± 3.07 efg 22.22 ± 2.07 def 28.77 ± 4.89 dg 24.45 ± 2.33 efg 28.23 ± 3.05 fgh 33.08 ± 2.89 h
p-Coumaric acid DL 18.95 ± 3.89 cde 28.55 ± 4.12 gh 16.10 ± 0.78 bcd 16.23 ± 0.77 bcd 10.83 ± 1.02 ab 20.24 ± 0.33 cde 31.08 ± 4.23 h 44.35 ± 2.67 i
L 11.19 ± 2.03 ab 15.80 ± 2.98 bc 10.31 ± 0.04 a 8.60 ± 1.23 aA 10.36 ± 0.38 ab 11.87 ± 0.99 ab

Abbreviations: D, darkness; DL, 12-h illumination and 12-h darkness photoperiod; L, constant illumination.
Means followed by different small letters are significantly different at ˛ < 0.05.
 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95 91

significant. Data was evaluated using Pearson’s correlation coef-

0.20 ± 0.04 abc

ficients to identify relationships between antioxidant activities,

0.17 ± 0.01 ab
1.52 ± o.07 cd

3.61 ± 0.14 ij
2.85 ± 0.43 e
2.99 ± 0.05 e

0.52 ± 0.10 e
12.02 ± 2.11 a
10.65 ± 1.07 a

14.56 ± 0.34 a

4.16 ± 0.78 f

3.68 ± 0.63 j
phenolics contents and selected antioxidant activities during
sprouts cultivation.

3. Results

0.94 ± 0.04 abc

0.23 ± 0.06 abc

3.1. Phenolics content in sprouts

19.77 ± 1.52 ab
22.30 ± 1.04 ab

0.27 ± 0.04 cd
3.16 ± 0.43 hi
1.64 ± 0.12 d

0.50 ± 0.09 e
16.15 ± 0.78 a

1.28 ± 0.11 c
4.10 ± 0.33 f

2.47 ± 0.32 f
Generally, different light condition during germination did not
influence the biomass production with the exception of days 6 and
8. Significant reduction in the fresh weight was observed in 6- and

6
8-days-old sprouts cultivated under light condition (Fig. 1).

39.23 ± 2.33 bcd

0.25 ± 0.02 bcd

In the investigated samples, there were important qualitative

45.04 ± 3.89 cde

0.19 ± 0.01 abc

29.68 ± 2.01 abc

1.86 ± 0.12 de
0.62 ± 0.04 ab

2.33 ± 0.01 ef
1.83 ± 0.06 d
0.53 ± 0.05 a
5.15 ± 0.78 g

0.73 ± 0.07 f
and quantitative differences in the phenolic composition of the
sprouts. The general trend of phenolics in lentil sprouts was a steady
decline during germination (Table 1). The influence of illumination
on the phenolics content and composition was clearly visible from

4
second day of cultivation. After 4 and 6 days of cultivation in the
presence of continuous light, the content of total phenolics, phe-

58.08 ± 7.63 defg

0.86 ± 0.23 abc

0.81 ± 0.04 abc

0.18 ± 0.03 abc

53.62 ± 2.78 def
53.19 ± 1.98 def
nolics acids and flavonoids in the sprouts increased markedly in

0.24 ± 0.03 bc
1.69 ± 0.11 cd

0.34 ± 0.06 d
6.76 ± 0.23 h

2.43 ± 0.34 f
2.41 ± 0.04 f
comparison to sprouts cultivated in darkness (Table 1). The level of
phenolic acids in sprouts obtained in the darkness and continuous
light was significantly lower in comparison to those from deter-
mined in sprouts cultivated in photoperiod. During the first 3 days

3
of cultivation, the level of condensed tannins was the highest in

59.15 ± 4.12 defg

1.20 ± 0.03 bcd

0.95 ± 0.23 abc

0.76 ± 0.12 abc

0.20 ± 0.02 abc

0.21 ± 0.01 abc

64.33 ± 3.89 efg

79.11 ± 1.78 gh

3.01 ± 0.43 gh

0.16 ± 0.04 ab
sprouts cultivated in darkness (Table 1).

2.59 ± 0.23 fg

2.64 ± 0.18 fg
The HPLC results indicated the presence of eleven hydrox-
ycinnamic and hydroxybenzoic acids and four flavonoids. It was
found that in dormant seeds, benzoic, sinapic and chlorogenic Time of germination (days)
were dominant components of phenolic acids fraction. The dom- 2
inant flavonoids fraction in the lentil seeds and seedlings was

0.88 ± 0.01 abc

0.22 ± 0.05 abc

0.18 ± 0.03 abc

(+)-catechin but the level of this compound was extremely reduced

0.17 ± 0.02 ab
1.22 ± 0.01 bc
98.71 ± 7.89 hi
113.25 ± 13.78 i
70.60 ± 2.89 fg

1.64 ± 0.08 d
0.50 ± 0.01 a

1.28 ± 0.23 c
1.34 ± 0.09 c
during germination (Table 3). The contents of the salicylic acid and
luteolin in the sprouts were significantly higher than in raw lentil

Abbreviations: D, darkness; DL, 12-h illumination and 12-h darkness photoperiod; L, constant illumination.
seeds but in other phenolics case there were not any simple rela-
Flavonoids composition of lentil sprouts cultivated under different illumination conditions (D, DL, L).

tionships (Tables 2 and 3). In the other cases various conditions of

1

germination led to significant influence on the content of phenolic

acids and flavonoids. It should be noted that cultivation with con-
1.23 ± 0.01 cd
138.75 ± 13.48 j

0.09 ± 0.01 a

0.14 ± 0.03 a
tinuous illumination (L) (in comparison to those sprouted in dark
conditions) increased the p-hydroxybenzoic, caffeic, gallic, salicylic
and o-coumaric acids content on days 3 and 4 after germination,
Soaked

the normal stage of lentil sprout consumption in the diet. Contrary

Means followed by different small letters are significantly different at ␣ < 0.05.
to these results, in the sprouts obtained in darkness, there was a
marked elevation in ferulic and p-coumaric acids levels, 3- and 2.2-
0.25 ± 0.02 bcd

fold increase above the sprouts cultivated in light (L), respectively

393.79 ± 20.75 k

0.74 ± 0.03 b
3.00 ± 0.19 e

(Table 2). Additionally, the level of chlorogenic acid was elevated

at a higher degree in sprouts cultivated with photoperiod (Table 2).
As it could be observed, 6- and 8-days-old sprouts obtained in pho-
toperiod were an excellent source of ferulic, benzoic, o-coumaric,
Raw

p-coumaric acids and luteolin. In the ready to eat sprouts (days

3–6) the highest quercetin content was recorded in the continu-
Light conditions

ously illuminated sprouts, while the lowest in those obtained in

the darkness (Table 3).
DL

DL

DL

DL

3.2. Antioxidant activities of sprouts

D

D
L

L
Compounds (␮g g−1 FM)

In order to analyze the effect of qualitative and quantitative

changes in the sprouts phenolics profile on the nutraceutical poten-
tial of this kind of food, the antioxidant capacities of sprouts were
determined (Figs. 2–5). The general trend of free radical scavenging,
(+)-Catechin

metal chelating and reducing properties in lentil sprouts showed

Quercetin

Daidzein
Luteolin

a steady decline with germination (Figs. 2–4). Radical scavengers

Table 3

of lentil acted in a dose-dependent manner (data not shown),

thus calculation of the 50% inhibitory concentration (IC50 ) was
92 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95

Fig. 2. Antiradical activity (ABTS) of lentil sprouts cultivated under different illumination conditions (D, DL, L). Abbreviations: D, darkness; DL, 12-h illumination and 12-h
darkness photoperiod; L, constant illumination. Means followed by different small letters are significantly different at ˛ < 0.05.

possible. It should be indicated that on days 4, 6 and 8 the sam- the lowest in those obtained on days 6 and 8 in the darkness (0.04
ples obtained under continuous light presented the highest abilities and 0.05 ␮g EDTA mg−1 FM, respectively) (Fig. 4). Germination with
to quench free radicals with respect to samples cultivated under illumination (D, DL) significantly increased the ability of sprouts to
darkness and darkness-illumination. The IC50 values determined prevent lipids peroxidation. The best results were obtained on days
for these samples were 4.45, 5.12 and 8.13 (mg ml−1 FM), respec- 1 and 2, however in sprouts on days 3 and 4, the stage of lentil sprout
tively (Fig. 2). Taking into account ready-to-eat lentil sprout (3-, 4-, consumption in the diet, the studied activity was high (Fig. 5). The
6-days-old) the highest antiradical potential was determine for 3- statistical analysis confirmed that the antioxidant activities, except
days-old sprouts. It should be noted that different light condition lipid prevention, were correlated significantly and positively with
during germination significantly influenced the reducing potential phenolics contents. Syringic, chlorogenic and p-hydroxybenzoic
of sprouts. Significant decrease in reducing abilities on days 4, 6, and acids and (+)-catechin are probably major contributors to these
8 were observed in sprouts cultivated at photoperiod (DL), while antioxidant activities based on the analysis of correlations. These
the highest abilities were recorded with continuously illuminated compounds are presented in the studied sprouts in high amounts;
(Fig. 3). From days 1 to 3, the ability to chelate metal ions was additionally reducing power, chelating and radical scavenging abil-
significantly elevated in cultivation performed with illumination ities were significantly and positively correlated with their level.
(constant light and photoperiod) in comparison to those obtained in Negative correlation between ability to inhibit the linoleic acid
darkness. The highest chelating power was determined on day 1 for peroxidation and others antioxidant abilities in spouts obtained in
continuously illuminated sprouts (0.26 ␮g EDTA mg−1 FM), while cultivation performed with illumination, can suggest that probably

Fig. 3. Reducing power (RP) of lentil sprouts cultivated under different illumination conditions (D, DL, L). Abbreviations: D, darkness; DL, 12-h illumination and 12-h darkness
photoperiod; L, constant illumination. Means followed by different small letters are significantly different at ˛ < 0.05.
 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95 93

Fig. 4. Chelating power (CHP) of lentil sprouts cultivated under different illumination conditions (D, DL, L). Abbreviations: D, darkness; DL, 12-h illumination and 12-h
darkness photoperiod; L, constant illumination. Means followed by different small letters are significantly different at ˛ < 0.05.

different classes of phytochemicals are involved in those activities Data concerning polyphenolics profile of dormant lentil seeds
(Table 4). are in agreement with those obtained in several previous stud-
ies (Amarowicz et al., 2010; Ghavidel and Prakash, 2007; Dueñas
et al., 2002, 2007). Phenolics compounds level in the lentil seedlings
4. Discussion
decreased during plant growth. A significant statistical reduction
was also observed in the total phenolic, tannin and flavonoid
Nowadays, one of the main areas of research is the search for
contents of black soybeans (Lin and Lai, 2006), soybean, lentil,
new food products that in spite of nutritional quality also pos-
alfalfa (Cevallos-Casals and Cisneros-Zevallos, 2010) and cowpea,
sess natural ingredients with biological activity e.g., antioxidant,
lentil, chickpea and green gram (Ghavidel and Prakash, 2007)
antiviral, antihypertensive, etc. (Siró et al., 2008).
due to the effect of germination. Losses may result from leach-
Plant phenolic metabolites are gaining interest due to their
ing into the soak water, binding of polyphenols with other organic
potential role in human disease prevention and treatment (Dixon,
substances such as carbohydrate or protein (Khandelwal et al.,
2001; Fang et al., 2002). These metabolites are mainly produced to
2010). Some variations of tannins content could be also due to
protect plants from biotic/abiotic stresses such as photooxidation
the activation of endogenous enzymes such as hydroxylases and
stress, reactive oxygen species, wounds, disease and herbivores
polyphenoloxydases whose activity increases during germination
(Shetty, 2004). Under normal conditions, during germination, lentil
(Matuschek and Svanberg, 2002; Udayasekhara Rao and Deosthale,
produces various secondary metabolites. Additionally, application
1987). The growth conditions of plants could affect the accumu-
of different cultivation conditions can result in an enhanced pro-
lation of antioxidant in response to different biotic and abiotic
duction of the antioxidants.

Fig. 5. Lipids peroxidation inhibition (LPI) of lentil sprouts cultivated under different illumination conditions (D, DL, L). Abbreviations: D, darkness; DL, 12-h illumination
and 12-h darkness photoperiod; L, constant illumination. Means followed by different small letters are significantly different at ˛ < 0.05.
94 M. Świeca et al. / Scientia Horticulturae 140 (2012) 87–95

Table 4
Pearson’s correlations among phenolics content and antioxidant activities (ABTS, RP, CHP, LPI) in lentil sprouts during germination under different illumination conditions
(D, DL, L).

ABTS RP CHP LPI

D DL L D DL L D DL L D DL L

Gallic acid 0.74 0.68 0.76 0.98 0.98 0.93 0.98 0.95 0.98 0.42 −0.34 −0.05
p-Hydroxybenzoic acid 0.69 0.61 0.17 0.97 0.89 0.58 0.97 0.76 0.97 0.33 −0.17 −0.72
Caffeic acid −0.47 −0.53 −0.77 0.12 0.02 −0.14 0.16 −0.03 0.16 −0.65 −0.47 −0.53
Benzoic acid −0.46 −0.60 −0.38 0.18 0.05 0.09 0.21 −0.05 0.21 −0.33 −0.25 −0.39
Syringic acid 0.64 0.51 0.61 0.54 0.59 0.57 0.43 0.33 0.43 0.18 −0.63 −0.38
Salicylic acid −0.83 −0.91 −0.84 −0.87 −0.77 −0.67 −0.80 −0.77 −0.80 −0.43 0.23 −0.16
Sinapic acid 0.53 0.53 0.54 0.96 0.94 0.96 0.96 0.88 0.96 0.30 −0.33 −0.31
Chlorogenic acid 0.88 0.91 0.68 0.94 0.94 0.94 0.91 0.87 0.91 0.49 −0.13 −0.36
Ferulic acid −0.66 0.65 0.53 −0.66 −0.25 0.57 −0.67 −0.33 −0.67 −0.80 −0.64 −0.43
o-Coumaric acid 0.23 −0.09 −0.01 −0.34 −0.45 −0.44 −0.43 −0.55 −0.43 −0.02 0.28 0.08
p-Coumaric acid −0.66 −0.65 0.53 −0.66 −0.25 0.57 −0.67 −0.33 −0.67 −0.80 −0.64 −0.43
(+)-Catechin 0.71 0.70 0.68 0.99 0.99 0.99 1.00 0.93 1.00 0.44 −0.23 −0.23
Quercetin −0.31 0.09 −0.32 0.23 0.50 −0.16 0.27 0.53 0.27 −0.52 −0.40 −0.24
Luteolin −0.35 −0.79 −0.83 −0.57 −0.76 −0.70 −0.53 −0.60 −0.53 0.14 0.11 0.11
Daidzein −0.20 −0.55 −0.30 0.15 −0.26 −0.24 0.21 −0.31 0.21 0.17 −0.24 −0.07
TPC 0.75 0.72 0.67 0.99 0.99 0.98 0.98 0.93 0.98 0.52 −0.19 −0.18
TFC 0.82 0.85 0.73 0.97 0.90 0.98 0.96 0.93 0.96 0.56 0.05 −0.13
PAC 0.71 0.65 0.64 0.99 0.87 0.99 0.98 0.97 0.98 0.46 0.09 −0.19
CTC 0.77 0.82 0.73 0.99 0.99 0.99 0.99 0.90 0.99 0.46 −0.34 −0.29
ABTS – – – 0.70 0.73 0.68 0.70 0.74 0.70 0.74 0.02 0.30
RP 0.70 0.73 0.68 – – – 0.99 0.91 0.99 0.43 −0.32 −0.30
CHP 0.70 0.74 0.81 0.99 0.91 0.91 – – – 0.45 −0.11 0.06
LPI 0.74 0.02 0.30 0.43 −0.27 −0.30 0.45 −0.14 0.45 – – –

Abbreviations: D, darkness; DL, 12-h illumination and 12-h darkness photoperiod; L, constant illumination. CTC, condensed tannin content; PAC, phenolic acids content; TFC,
flavonoids content; TPC, total phenolics content; ABTS, radical scavenging ability; CHP, chelating power; LPI, inhibition of linoleic acid peroxidation; RP, reducing power.

stresses (Shetty, 2004; McCunea and Johns, 2007; Gawlik-Dziki from conjugated glycosides due to the enzymatic activation (Lin
et al., 2012). Data provided by these studies showed that reduction and Lai, 2006). Lentil sprouts with stronger reducing power, chelat-
of total polyphenols concentrations due to germination differed ing power and ability to quench free radical had higher amounts
significantly between studied conditions of cultivation. Levels of of total phenolics, flavonoids and condensed tannin contents with
polyphenols determined in sprouts suggest that too strong illumi- good coefficient of determination. On the other hand, the corre-
nation may induce overproduction of antioxidants due to oxidative lations between total phenolics and lipid prevention abilities in
stress. These data are in agreement with previous reports by sprouts cultivated with light were negative which suggests that in
Khattak et al. (2007) and Shetty et al. (2002). Cited investigators this case an increase of activity was not due to subsequent elevation
proved that different illumination conditions might induce the of phenolic antioxidant content.
proline-linked pentose phosphate pathway for phenolics synthesis.
Correlation between illumination intensity and salicylic acid con-
tent in sprouts may confirm the appearance of stress and the role 5. Conclusion
of this compound in signal transduction pathway (Yuan and Lin,
2008). Lentil sprouts were a very rich dietary source of p-coumaric, In conclusion, it should be noted that lentil sprouts are a good
ferulic and caffeic acids. It might be speculated that in sprouts culti- source of polyphenolic compounds and have significant antioxi-
vated under continuous light, elevation in the levels of these lignin dant potential. Additionally, data provided by this work confirmed
biosynthesis intermediates was linked with cross-talk response on a key role of cultivation condition on the chemical composition
stress conditions (Solecka, 1997; Fujita et al., 2006). Additionally, and potential biological activity of sprouts. The illumination causes
high levels of p-coumaric acid might be involved in overproduction changes in phenolics profile which are translated to a significant
coumarylCoA, a key compound in flavonoids biosynthesis (Zabala improvement of antioxidant potential of low-processed sprouted
et al., 2006). food.
Germination as well as illumination condition significantly
modified the antioxidant potential of sprouts. Free radical scaveng-
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