Antioxidant and Antihypertensive Potential of Protein Fractions from Flour and Milk Substitutes from Canary Seeds (Phalaris canariensis L.)

Abstract

Canary seed (Phalaris canariensis) is used to feed birds but it has been recently considered a promising cereal with nutraceutical potential for humans. The aim of this work was to analyze the protein fractions from canary seed flour and from milk substitutes (prepared by soaking the seeds in water 12 and 24 h), and to evaluate antioxidant and antihypertensive capacity of peptides obtained after in vitro digestion. Prolamins were the major protein fraction, followed by glutelins. After digestion, albumins and prolamins fractions from milks presented higher levels of peptides than flour, globulins showed more peptides in flour and glutelins were found in similar concentrations in all samples; 24 h milk prolamins had the highest concentration of peptides. Purification by high performance liquid chromatography (HPLC), sequencing of peptides, in vitro antioxidant ABTS (2,2'-azino-bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, and antihypertensive capacity (angiotensin converting enzyme (ACE) assay), indicated that peptides from canary seed prolamins were the most efficient compounds with antioxidant and antihypertensive activity. Canary seeds may be considered an accessible and cheap source to prepare milk substitutes with high contents of bioactive peptides with remarkable functional properties to promote better human health and healthy ageing.

Full text

ORIGINAL PAPER
Antioxidant and Antihypertensive Potential of Protein
Fractions from Flour and Milk Substitutes from Canary
Seeds (Phalaris canariensis L.)
María Elena Valverde
1
&Domancar Orona-Tamayo
1
&Blanca Nieto-Rendón
1
&
Octavio Paredes-López
1
#Springer Science+Business Media New York 2016
Abstract Canary seed (Phalaris canariensis)isusedtofeed
birds but it has been recently considered a promising cereal
with nutraceutical potential for humans. The aim of this work
was to analyze the protein fractions fromcanary seed flourand
from milk substitutes (prepared by soaking the seeds in water
12 and 24 h), and to evaluate antioxidant and antihypertensive
capacity of peptides obtained after in vitro digestion.
Prolamins were the major protein fraction, followed by
glutelins. After digestion, albumins and prolamins fractions
from milks presented higher levels of peptides than flour,
globulins showed more peptides in flour and glutelins were
found in similar concentrations in all samples; 24 h milk pro-
lamins had the highest concentration of peptides. Purification
by high performance liquid chromatography (HPLC), se-
quencing of peptides, in vitro antioxidant ABTS (2,2′-azino-
bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-
diphenyl-1-picrylhydrazyl) assays, and antihypertensive ca-
pacity (angiotensin converting enzyme (ACE) assay), indicat-
ed that peptides from canary seed prolamins were the most
efficient compounds with antioxidant and antihypertensive
activity. Canary seeds may be considered an accessible and
cheap source to prepare milk substitutes with high contents of
bioactive peptides with remarkable functional properties to
promote better human health and healthy ageing.
Keywords Nutraceuticals .Antihypertensive .Antioxidants .
Canary seed proteins .Bioactive peptides
Introduction
Phalaris canariensis L., commonly known as canary seed, is
an annual cereal crop originating in the Mediterranean region;
moreover, it is mainly cultivated in Argentina and Canada.
Now, it has been considered a promising cereal for human
consumption due to its excellent nutritional and nutraceutical
value, as well as for food industrial purposes [1].
Robinson [2,3] was the first to report the food potential of
canary seed; he also found no toxic characteristics for human
consumption. Abdel-Aal et al. [4–6] described that canary
seed has 19–24 % protein, 5–9 % crude fat, 6–8 % total dietary
fiber, 2 % ash, and 55–61 % starch. Putnam et al. [7,8]re-
ported a high mineral content in the seeds, and Abdel-Aal
et al. [5] found that they have some B complex vitamins, such
as thiamine, niacin and riboflavin. The oil is highly unsaturat-
ed, including linoleic (55 %), oleic (29 %), and linolenic
(2.5 %) acids; it is worth mentionining that the seed has shown
excellent antioxidant capacity [4]. P. canariesis seed also con-
tains high levels of protein compared with other cereal grains,
the proportion of prolamins and glutelins fractions is 78 %;
they are rich in cysteine, tryptophan, and phenylalanine, but
deficient in lysine and threonine [4,9]. Boye et al. [10]
reported the absence of gluten in canary seed, thus it can
be included in gluten-free diets for people with celiac
disease; besides, no cross-reaction was detected using
ELISA between canary seed proteins and almond, hazelnut,
mustard, peanut, sesame, soybean and walnut proteins.
The potential benefits of canary seeds in human health
has been widely used in folk medicine. They have shown
anti-hypertensive, anti-diabetic and anti-inflammatory effects,
Electronic supplementary material The online version of this article
(doi:10.1007/s11130-016-0584-z) contains supplementary material,
which is available to authorized users.
*María Elena Valverde
malevalverde@gmail.com
1
Centro de Investigación y de Estudios Avanzados de Instituto
Politécnico Nacional, Km. 9.6 Libramiento Norte Carretera
Irapuato-León, CP 36821 Irapuato, Guanajuato, Mexico
Plant Foods Hum Nutr
DOI 10.1007/s11130-016-0584-z

blood lipid and cholesterol lowering capacity, and have been
also described as remedy for kidney, pancreas, bladder dis-
eases, and obesity [9–14]. Their use in human health is in-
creasing due to their antihypertensive effects, inhibition of
urinary tract infections, arteriosclerosis, AIDS, gout, rheuma-
tism, edema, gastritis and stomach ulcer; also provide muscle
tone and prevent cardiovascular disorders [15].
P. canariensis is commonly used as a remedy known
Bcanary seed milk substitutes or milk^[15], which is prepared
by soaking the seeds in water for a period of 12 or 24 h then
the seeds are ground with clean water. It has been usually
described as nutraceutical agent for human health; however,
no scientific information has been reported regarding these
seed milks. The aim of this study was to analyze the protein
fractions from canary seed flour, and milk obtained by a tra-
ditional process, in order to determine their antioxidant and
antihypertensive potential.
Materials and Methods
Sample Preparation Canary seed was provided by local pro-
ducers (Irapuato, Gto., Mexico). Seeds were ground in a mill
(Brinkmann Retsch, USA) and passed through an 80 mesh to
obtain the flour. For the milk substitutes, 5 g of seeds were
soaked in 250 mL of distilled water for 12 and 24 h at room
temperature. The soaking water was removed and the seeds
were milled in an Osterizer blender with 250 mL fresh dis-
tilled water. The mixture was percolated through a sieve and it
was lyophilized (FreeZone, 2.5, Labconco) and stored at
−20 °C, until use.
Extraction of Protein Fractions Proteins from flour and
milks were fractionated as reported by Sandoval-Oliveros
and Paredes-López [16]; the protein concentration was
determined with a Pierce BCA protein assay kit
(Thermo Scientific, USA).
Polyacrylamide Gel Electrophoresis The first electrophoret-
ic separation of protein and peptides was performed by 13 and
14 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
For two-dimensional gel electrophoresis (2D–PAGE) was
used a 2D cleanup kit (GE Life Sciences, Sweden) following
manufacturer’s instructions. Proteins were loaded on 7 cm
IPG dry strip and were focused according to manufacturer’s
instructions (GE Life Sciences, Sweden).
In Vitro Digestion of Protein Fractions A sequential enzyme
digestion was carried out following Wang et al. [17] procedure.
ACE Assay The inhibitory effects of canary seed peptides
against ACE were determined according to studies carried
out in our laboratory by Luna-Suárez et al. [18] and Orona-
Tamayo et al. [19].
ABTS Radical-Scavenging Activity This assay was per-
formed according to the method by Zhou et al. [20], with
modifications by Orona-Tamayo et al. [19].
DPPH Radical-Scavenging Activity This assay was mea-
sured according to Martínez-Cruz and Paredes-López [21]
procedure, modified by Orona-Tamayo et al. [19].
Peptides Separation by HPLC The fractionation of flour
prolamins was performed following the method described by
Orsini-Delgado et al. [22] using a preparative C-18 HPLC
column on a HPLC System (Waters, Milford, MA). The main
portion was collected at 9.85 min and then freeze-dried and
stored at −70 °C until use.
Peptide Identification by Liquid Chromatography-Mass
Spectrometry (LC-MS) Analysis Freeze-dried flour prola-
min peptides were applied in a LC-MS system according to
Meneses et al. [23] procedure. The MS spectra data were
collected in positive ion mode and subjected to identification
with Proteome Discoverer 1.4 and BLAST. The functional
prediction of prolamin bioactive peptides was analyzed using
BIOPEP database [24].
Statistical Analysis Experiments were performed with four
repetitions and results were expressed as means ± standard
deviation and analyzed using global LSD (least significant
difference) and univariate analysis of variance (ANOVA).
Statistical analyses were carried out using Statistical Package
for the Social Sciences 17.0 (SPSS Inc., Chicago, IL).
Results and Discussion
Protein Fractions The major protein fraction in canary seeds
was prolamins (Table 1,Online supplementary material):
54 % in flour, 46.3 % in 12 h milk and 38.1 % in 24 h milk.
Tabl e 1 Angiotensin converting enzyme -inhibitory activity (IC
50
)
Milk substitutes
Fractions/Samples Flour 12 h 24 h
Albumins 504.8 ± 82
a
450.1 ± 85
a
500.5 ± 94
a
Globulins 442.8 ± 68
a
384.1 ± 62
a
483.6 ± 89
a
Prolamins 217.4 ± 18
a
276.4 ± 21
b
279.8 ± 13
b
Glutelins 348.9 ± 45
a
396.7 ± 80
a,b
518.6 ± 69
b
Different letters in the same row indicate significant differences (P≤0.05)
IC
50
=μgpeptides/mL
Plant Foods Hum Nutr

Prolamins are the main protein in maize, wheat, barley and
rye; they are determinant for kernel texture in the mature seed,
an essential characteristic of cereal grains [25]. The second
most abundant fraction was glutelins: 26.6 % in flour,
19.7 % in 12 h milk and 12.2 % in 24 h milk. The amount
of globulins was very similar in flour and milks, but albumins
showed significant variations: 4.8 % in flour, 17.3 % in 12 h
milk and 32.8 % in 24 h milk. The protein fraction values were
relatively similar to some previous studies in canary seed flour
[4,9]. The protein concentration changed in 12 and 24 h milk
samples compared with flour; it may be due to the digestion or
degration ofproteins when soaking the seeds in water; further-
more, prolamins were the main fraction in all samples ana-
lyzed (Table 1,Online supplementary material).
Electrophoretic Pattern of Protein Fractions Except for
globulins, there were no significant differences in the electro-
phoretic pattern in flour and milk samples. Albumins present-
ed diffused bands of low molecular weight (20 to 100 kDa)
and clear bands between 35 to 45 kDa; milks showed one
band of around 100 kDa (Fig. 1a). Globulins had bands
around 12 to 20 kDa , and others more clear around 40 to
60 kDa; 12 and 24 h milks exhibited a band of 60 kDa and
an interestingly reduction was observed in a single band
around 37 kDa. Prolamins showed two bands of 10 and
22 kDa approximately; profile which is similar to oat, maize,
rice and barley [25]. An apparent proteolysis in the main band
was found only in 24 h milk. In glutelins, some bands were
located between 25 to 35 kDa and 40 to 60 kDa; a dense band
of approximately 40 kDa appeared in 24 h milks, which may
result by a degradation effect (Fig. 1a). Our results from flour
were similar to those found by Estrada-Salas et al. [9]; but
when canary seeds were soaked in water for milk preparation,
the imbibition period induces proteolysis of some proteins and
electrophoretic pattern changes, which promote the release of
peptides. Up to our knowledge this is the first time that 2D–
PAGE electrophoresis is reported in canary seeds. All the
fractions were well separated by SDS-PAGE and 2D–PAG E
and some similarities in electrophoretic patterns in all fractions
were observed (data no shown). By SDS-PAGE, prolamins
were detected mainly between 15 and 20 kDa and one band
around 50 kDa; by 2D–PAGE, the range of isoelectric point
was rather wide [3,10]. As indicated above, profiles were
similar between flour and milk substitutes; but it was clear a
protein degradation in milks, especially at 24 h (Fig.1b).
Canary Seed Proteins Digestion A large variety of benefits
appear when proteins are digested in the gastrointestinal tract
and thus bioactive peptides are released from food. Canary
seed proteins have been digested more easily under sequential
digestion [26]; our digestion method appears to be appropriate
because after 6 h of in vitro digestion all protein fractions were
completely degraded (Fig. 1a, Online supplementary
material). Estrada-Salas et al. [9] reported similar results in
flour. Albumins and prolamins from milks released more pep-
tides than the same protein fraction from flour, and prolamins
from 12 h milk presented the highest peptides concentration,
Fig. 1 SDS and 2D–PAGE from canary seeds protein fractions from
flour and milk subtitutes. Mmolecular marker, Fflour, 12 12 h milk,
24 24 h milk, Ip isoelectric point
Fig. 2 Free radical scavenging activity from canary seeds flour and milk
subtitutes. Fflour, 12 12 h milk, 24 24 h milk
Plant Foods Hum Nutr

followedby12and24halbumins(Fig.1b, Online
supplementary material). Flour globulins released more pep-
tides than the same fraction in milk samples; however,
glutelins showed statistically (p≤0.05) the same proportion
of peptides in all samples. These results indicate that prola-
mins and albumins generate more peptides after digestion,
which suggest their susceptibility to proteolysis (Fig. 1b,
Online data). Interestingly, when the canary seed is soaked
in water a natural pre-digestion occurs, thus facilitating the
release of peptides from the protein fractions.
ACE Activity of Canary Seed Peptides Food peptides can
be considered an effective and safe strategy against high blood
pressure [27]; peptides from flour and milks were evaluated
for their antihypertensive action. Prolamins peptides showed
more inhibition than peptides from the other fractions, espe-
cially flour with an IC
50
of 217.4 μg/mL; moreover, it was not
statistically (p≤0.05) different to globulins and glutelins from
12 h milk and glutelins from flour (Table 1). No effect was
found against angiotensin activity when soaking canary seeds
for 24 h, with exception of prolamins; however, soaking for
12 h considerably increased their activity. Estrada-Salas et al.
[9]reported an IC
50
value of 332 μg/mL for canary seeds
flour peptides, similar to other seeds and less effective to
our results. Passos et al. [14] reported that the aqueous
extract of canary seeds reduced systolic blood pressure
in hypertensive Wistar rats; the interruption of the treatment
caused a rise in the blood pressure. They reported no negative
Tabl e 2 Peptides identified by
LC-MS in canary seed prolamin
fractions from 12 h milk
substitute
Peptide Molecular weight (Da) Peptide-sequences ID Activity function
LSLGT 664.36 GT 7612 ACE inhibitor
•LG 7619 ACE inhibitor
•SL 7619 Dipeptidyl-peptidase IV inhibitor
••••
TDQPAG 703.38 AG 7600 ACE inhibitor
•PA 3179 Dipeptidyl-peptidase IV inhibitor
•QP 8532 Dipeptidyl-peptidase IV inhibitor
•AG 8760 Dipeptidyl-peptidase IV inhibitor
•DQ 8768 Dipeptidyl-peptidase IV inhibitor
•TD 8898 Dipeptidyl-peptidase IV inhibitor
••••
QQLQT 797.42 LQ 7831 ACE inhibitor
•QL 8874 Dipeptidyl-peptidase IV inhibitor
•QQ 8876 Dipeptidyl-peptidase IV inhibitor
•QT 8878 Dipeptidyl-peptidase IV inhibitor
FEPLQLA 926.5 PL 7513 ACE inhibitor
•LA 7585 ACE inhibitor
•LQ 7831 ACE inhibitor
•LA 3175 Dipeptidyl-peptidase IV inhibitor
•EP 8529 Dipeptidyl-peptidase IV inhibitor
•PL 8638 Dipeptidyl-peptidase IV inhibitor
•QL 8874 Dipeptidyl-peptidase IV inhibitor
KPQLYQPF 1019.52 LY 3381 ACE inhibitor
•KP 7810 ACE inhibitor
•PQ 7837 ACE inhibitor
•LY 7872 Antioxidant
•KP 8218 Antioxidant
•KP 8519 Dipeptidyl-peptidase IV inhibitor
•QP 8532 Dipeptidyl-peptidase IV inhibitor
•PF 8854 Dipeptidyl-peptidase IV inhibitor
•PQ 8861 Dipeptidyl-peptidase IV inhibitor
•QL 8874 Dipeptidyl-peptidase IV inhibitor
YQ 8943 Dipeptidyl-peptidase IV inhibitor
Bioactive peptides were obtained from BIOPEP database (http://www.uwm.edu.pl/biochemia/) [24]
Plant Foods Hum Nutr

side effects in renal function, diuresis or Na
+
excretion. Our
results showed that proteins from canary seed are a good
source of peptides inhibitors of ACE, mainly prolamins.
Antioxidant Activity of Canary Seed Peptides The ABTS
assay showed different inhibition patterns (Fig. 2a). Albumins
and prolamins peptides of 12 h milk showed the best IC
50
values, 133.2 and 181.6 μg/mL, respectively (p≤0.05); also,
albumins and glutelins of 24 h milk showed high IC
50
values,
176.0 and 213.7 μg/mL, respectively (p≤0.05). Canary seed
flour presented the lowest IC
50
values in all protein fractions,
except for prolamins (Fig. 2a). These results clearly demon-
strate that the protein undergoes hydrolysis by soaking the
seeds in water, thus milk samples showed more antioxidant
potential than the flour in all protein fractions. Prolamins pep-
tides, in all cases, were the most efficient samples to inhibit the
DPPH activity (p≤0.05) comprising the lowest IC
50
(Fig. 2b).
The 24 h milk showed the best antiradical IC
50
value (89.7 μg/
mL) followedby flour and 12 h milk (104.2 and 114.1 μg/mL,
respectively). Albumins and globulins peptides showed a sim-
ilar trend against DPPH; milks were more efficient than the
seed flour. Glutelins peptides presented similar IC
50
values:
flour 248.5, 12 h milk 254, 24 h milk 253.2. In general, 24 h
milk tended to show better inhibition against DPPH in
almost all fractions (albumins: 144.7; globulins: 147.6;
prolamins: 89.7 μg/mL), except for glutelins. Albumins
and globulins from milks showed more antioxidant capacity
than the flour, whereas prolamins and glutelins did not follow
the same trend.
Peptides Separation and Identification HPLC chromato-
gram of prolamins peptides presented a main peak with a
retention time of 9.85 min (Fig. 2,Online supplementary
material); this peak was collected and peptides were analyzed
by LC-MS. Five peptides were identified in a molecular
weight range between 664 and 1019 Da (Table 2). Databank
revealed that some peptides had inhibitory activities against
dipeptidyl-peptidase IV (DPPIV) and ACE. In most cases,
dipeptides encrypted in KPQLYQPF, FEPLQLA and
TDQPAG sequences showed the highest proportion of
DPPIV (dipeptidyl-peptidase), ACE inhibitors and antioxi-
dants, respectively. Dipeptides LA and PA displayed an
inhibition of DPPIV activity [28]. It has been demonstrated
that these peptides of canary seeds showed 43.5 % of inhibi-
tion against DPPIV [9]. Both peptides were found in our se-
quences and can interact between DPPIV mainly via hydro-
phobic interactions. Peptides with GT, LG, LY, LA, LQ, AG,
PL, KP and PQ were associated to antihypertensive functions
with the potential to inhibit ACE [29]; these sequences
were found in canary seed prolamins. We also found two
dipeptides (LY and KP) that have been previously reported
with antioxidant activity [27]. Capriotti et al. [30] reported
peptides with ACE inhibitory activity and antioxidant capacity
in soybean flour and soy milk. Our results suggest that the
prolamins fraction of canary seeds may be considered an ex-
cellent source of peptides with inhibitory capacity against
DPPIV and ACE, as well as antioxidant.
Conclusion
Up to our knowledge, this is the first study for the determina-
tion of protein fractions and their nutraceutical potential of
canary seed milks. Prolamins were the most abundant fraction
in canary seed flour and milk substitutes. Peptides from canary
seed flour and milk substitutes showed a high potential as
antioxidants and ACE inhibitors; particularly prolamins frac-
tion from milk substitutes showed the strongest antihyperten-
sive capacity. It is worth mentioning that canary seed milk is
easy, cheap and fast to obtain. In brief, P. canariensis seeds
showed high nutraceutical levels with the high potential to
impact positively human health. Therefore, canary seed is a
promising cereal for human consumption in the years to come.
Acknowledgments We acknowledge partial support from Consejo
Nacional de Ciencia y Tecnología, México to carry out this study. The
authors wish to thank Talía Hernández Pérez for technical assistance,
CINVESTAV-Irapuato, México.
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict of interest. This
article does not contain any studies with human or animals subjects.
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