Journal of Cereal Science 69 (2016) 363e370

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Journal of Cereal Science

journalhomepage:www.elsevier.com/locate/jcs

Antioxidant activity, phenolic profile, chlorophyll and mineral matter content of

corn silk (Zea mays L): Comparison with medicinal herbs
a, * a c b d

SlaCana Zilic , Marijana Jankovic , Zorica Basic , Jelena Vancetovic , Vuk Maksimovic
a Maize Research Institute, Department of Food Technology and Biochemistry, Slobodana Bajica 1, Zemun, 11085 Belgrade, Serbia
b Maize Research Institute, Breeding Department, Slobodana Bajica 1, Zemun, 11085 Belgrade, Serbia
c Military Medical Academy, Institute of Hygiene, Crnotravska 17, 11000 Belgrade, Serbia
d Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, 11000 Belgrade, Serbia

article info abstract

Article history: Yellow, green, pinky and purple colored corn silk were harvested 5 and 25 days after emergence and compared with six
Received 30 November 2015 medicinal herbs for total phenolics, flavonoids, anthocyanins, proanthocyanidins, chlorophylls, as well as antioxidant activity.
Received in revised form 4 April The phenolic acid, flavonoid and mineral matter profiles of corn silks were also determined. Corn silks at the silking stage are
2016
much more suitable for use as a natural source of phenolic compounds than silks at the R4 dough stage. The content of total
Accepted 2 May 2016
phenolic and flavonoid compounds in fresh silk was higher by about 2- to 4-fold than in the silk at the R4 dough stage and by
Available online 3 May 2016
about 1.2- to 2.6-fold than in the most investigated herbs. The most abundant phenolic com-pounds were hydroxycinnamic
acid ester and luteolin derivative i.e. 3-O-caffeoylquinic acid and maysin, respectively. A high potassium content and
Keywords:
antioxidant activity of silk are associated with its health benefit properties.
Antioxidant activity
Corn silk
Medicinal herbs
Mineral matters © 2016 Elsevier Ltd. All rights reserved.
Phenolic profiles

1. Introduction
Written records about medicinal herbs and traditional methods of their use
in the treatment of diseases date back over five millennia. Natural products,
especially those derived from herbs, as parts of foods or plant potions,
decoction, essential oils and pow-ders have been used therapeutically to
provide proactive support to various physiological systems and to cure and
prevent diseases such as common cold, flu, digestive and/or intestinal
diseases, headache, stomach ulcer, nervousness, circulatory disorders,
bronchitis, skin diseases, and others (Gurib-Fakim, 2006). In spite of the great
advances observed in modern medicine in recent decades, according to report
of the World Health Organization, more than 70% of the world population,
particularly in the developing coun-tries, rely on non-conventional medicine
in their primary health-care (Akerele, 1993). Furthermore, medicinal plant
products have contributed enormously to the development of important thera-
peutic drugs used currently in modern medicine (Balunas and Kinghorn,
2005) due to their high concentration of phytochemi-cals that are well
documented for their pharmacological activity.
The common property of these compounds is antioxidative activity, but they
also possess a variety of specific properties.
Maydis stigma is also considered as one of the herbal drugs. It represents
the threads which make the female flower of Zea mays L. commonly known
as “corn silk”. Corn silk has a long history of use. Historically, corn silk has
been used for the treatment of urinary tract disorders, pathological swelling,
asthma, dropsy and hyper-tension (Wynn and Fougere, 2007). Traditional
medicine in many parts of the world such as China, Turkey, Serbia, United
States and France has been using corn silk for the treatment of cystitis, edema,
kidney stones, diuretic, prostate disorders, and urinary infections, as well as
bedwetting and obesity (Wynn and Fougere, 2007). Recent research shows
that corn silk promotes bile flow (Wynn and Fougere, 2007), causes inhibition
of IgE formation by glycoproteins, reduces hyperglycemia (Guo et al., 2009),
has neuroprotective ef-fects against oxidative stress (Choi et al., 2014) and
anti-fatigue activity in animals by inhibiting the production of blood lactic
acid (Hu et al., 2010), as well as antitumor effect (Yang et al., 2014). Potential
healthcare applications of corn silk are very much related to its chemical
composition and mechanism of action of its bioac-tive constituents such as
phenolics and terpenoids, as well as polysaccharides and glycoproteins.
Known for their antioxidant and free radical scavenging capacity,
flavonoids are major active

* Corresponding author.
E-mail address: szilic@mrizp.rs (S. Zilic).

http://dx.doi.org/10.1016/j.jcs.2016.05.003
0733-5210/© 2016 Elsevier Ltd. All rights reserved.
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S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370
phenolic compounds and ingredient in corn silk that possess various
pharmacological activities. Among others, maysin [rham-nosyl-6-C-(4-
0 0
ketofucosyl)-5,7,3 ,4 -tetrahydroxyflavone], a flavone glycoside containing a
rhamnose residue, is the most abundant flavonoid in corn silk (Ren et al.,
2009). Diuretic action of corn silk is thought to be in part to the high
þ individual flavonoids were extracted from 50 mg of corn silk by mixing with
concentration of K salt (Velazquezd et al., 2005). Corn silk also contains
2þ
proteins, vitamins, carbohydrates, Ca , Mg
2 þ þ
and Na salts, fixed and
volatile oils, steroids such as sitosterol and stigmasterol, alkaloids, saponins
and tannins.
Currently, in spite of various pharmacological activities, corn silk is still
considered as a waste from corn processing. Only in some countries, corn
silk-based products such as tea, powder, and cos-metics are commercially
available. Since there are the ample op-portunities to convert corn silk as
waste into value-added corn products, especially in countries with high corn
production, the aim of this study was to investigate the content of bioactive
compounds in purple, green, pinky and yellow corn silks. Corn silks were
compared with six medicinal herbs for total phenolics, flavonoids,
anthocyanins, proanthocyanidins, chlorophylls, as well as antioxi-dant
activity. In addition, the effect of harvest maturity on phenolic acid, flavonoid
and mineral matter profiles of corn silks were determined.
2. Materials and methods
2.1. Corn silk and medicinal herb samples
Four corn silk samples were collected from ears of hybrids, ZP Exp, ZP
555, ZP 341, ZP 366, developed at the Maize Research Institute Zemun Polje
(MRIZP) (Belgrade, Serbia). The genotypes were selected according to silk
color, which varied from purple, green, pinky and yellow in ZP Exp, ZP 555,
ZP 341 and ZP 366 hy-brids, respectively. Selected genotypes were grown
during 2015 in the field of the MRIZP in a randomized complete block design
(RCB) with two replications. Standard cropping practices were applied to
provide adequate nutrition and to keep the disease-free plots. The silks were
harvested 5 to 7 (silking stage-R1) and 25 to 27 (dough stage-R4) days after
their emergence. Fresh medicinal herbs (Mentha piperita, Melissa officinalis,
Ginkgo biloba, Thymus serpyl-lum, Salvia officinalis) were obtained from
local farmer. The leaves of these plants were harvested shortly before
flowering and used for analysis. The sample of Thymus serpyllum was
consisted of a mixture of leaves and flowers at full developmental stages.
Collected silks and medicinal herbs (about 500 g) were cleaned of impurities,
washed with distilled water and dried in the ventilated oven at room
temperature for 24e36 h. Dry green tea was pur-chased from a local
supermarket. The dried corn silks and medic-inal herbs were ground using a
Perten 120 lab mill (Perten, Sweden) into a fine powder (particle size <500
mm) and stored in a screwed cap bottle at 4 C.
2.2. Analytical procedures
2.2.1. Chemicals
All chemicals and solvents were of HPLC or analytical grade. Acetone,
sodium hydroxide, hydrochloric acid, ethanol, sodium carbonate, sodium
nitrite and aluminum chloride were purchased from Merck (Darmstad,
Germany). Formic acid and acetonitrile were purchased from J.T. Baker
(Deventer, Holland). Cation stan-dard solution was purchased from Fluka
Chemie AG (Buchs,
Switzerland). Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl
(DPPH), methanesulfonic acid, (þ)-catechin and chlorogenic acid were
purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was
used throughout the experiments.
2.2.2. Extraction of free phenolic compounds
For the detection of the DPPH scavenging activity, total phe-nolics, total
flavonoids and proanthocyanidins, extracts were pre-pared by continuous
shaking of 150 mg of grinded corn silk and medicinal herb samples in 10 ml
of 70% aqueous acetone for 1 h at room temperature. Phenolic acids and
10 ml of 95% methanol. After centrifugation (5 min at 14000 g and 4 C) su-
pernatants were used for experiments. All extractions were per-formed in
triplicate.
2.2.3. DPPH radical scavenging assay
The DPPH scavenging activity was determined according to the Abe et al.
(1998) assay. Briefly, an aliquot of extract (0.1 ml) was transferred into test
tube. The volume was adjusted to 0.5 ml with 70% acetone and mixed with
the DPPH reagent (0.5 mM in ethanol, 0.25 ml) and the acetate buffer (100
mM, pH 5.5, 0.5 ml). After standing for 30 min in the dark, the absorbance
was measured at 517 nm against a blank containing acetone instead of a
sample aliquot. The results were expressed as an IC 50 value that represents
the amount of grinded samples (in mg of d.m.) providing 50% in-hibition of
DPPH .
2.2.4. Analysis of total phenolic compounds
The total phenolic content was determined by the Folin-Ciocalteu assay
according to Singleton et al. (1999), and expressed as mg of chlorogenic acid
equivalent (CGAE) per 100 g of dry matter (d.m.). An aliquot (0.05 ml) of
aqueous acetonic extract was transferred into test tubes and their volumes
adjusted to 0.5 ml with distilled water. After addition of the Folin-Ciocalteu
reagent (0.25 ml) and 20% aqueous Na 2CO3 solution (1.25 ml), tubes were
vortexed. After 40 min, the absorbance was recorded at 725 nm.
2.2.5. Analysis of total flavonoids
The flavonoid content was determined according to Eberhardt et al.
(2000), and expressed as mg of catechin equivalent (CE) per 100 g of d.m.
Briefly, 0.05 ml of 5% NaNO2 solution was mixed with 0.1 ml of the extract.
After 6 min, 0.5 ml of 10% AlCl3 solution was added to form a flavanoid-
aluminum complex. After 7 min, 0.25 ml of 1 M NaOH was added, and after
10 min the mixture was centrifuged at 9000 g for 5 min. Absorbance of the
supernatant was measured at 510 nm.
2.2.6. Analysis of proanthocyanidins
Proanthocyanidins were determined by the butanol-HCl assay
described by Zilic et al. (2011). Briefly, 0.5 ml of the sample extract was
mixed with 3.0 ml of butanol-HCl reagent (95:5 v/v) and 0.1 ml ferric reagent
(2% ferric ammonium sulfate in 2.0 M HCl). After boiling for 60 min in a
water-bath, the absorbance was measured at 550 nm. The content of
proanthocyanidins was calculated as a leucocyanidin equivalent (LE)
according to the formula: (A550 nm 78.26 dilution factor)/(% dry weight).
2.2.7. Analysis of total anthocyanins
The anthocyanins content was determined according to method
described by Zilic et al. (2012), and expressed as mg of cyanidin 3-glucoside
equivalent (CGE) per 100 g of d.m. Grinded samples (300 mg) were extracted
by mixing with 10 ml of methanol acidi-fied with 1 M HCl (85:15, v/v) and
shaking for 30 min at ambient temperature. The crude extract was centrifuged
at 8000 g for 20 min and absorbances of supernatant at 535 and 700 nm were
measured to detect anthocyanins.
2.2.8. Analysis of phenolic acids and individual fl avonoids
Separation
of phenolic compounds was done by the reversed

S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370
phase HPLC analysis. Samples were injected in the Waters HPLC system
consisting of 1525 binary pumps, thermostat and 717 þ autosampler
connected to the Waters 2996 diode array and EMD 1000 single quadropole
detector with the ESI probe (Waters, Milford, USA). Separation of phenolics
was performed on a Sym-metry C-18 RP (Waters, Milford, MA, USA)
column 125 mm 4 mm with 5 mm diameter particles, connected to an
appropriate guard column. Two mobile phases, A (0.1% formic acid in water)
and B (acetonitrile) (J.T. Baker, Deventer, Holland) were used at a flow rate of
1 ml/min at 30 C with the following gradient profile: the first 30 min rise from
10 to 60% B; in next 10 min reverse to initial 10% B; followed by 5 min of
the equilibration time. The post column flow splitter (ASI, Richmond, CA,
USA) with 5/1 split ratio was used to obtain optimal mobile phase inflow for
the ESI probe. Peaks of detected compounds were located and identified in the
HPLC-UV chromatograms (recorded at 326 nm) through comparison of
separated standards with authentic samples by combining their retention times
and UV spectra obtained by DAD. For the LC/MS analysis, signals for each
compound were detected in a negative the ESI scan (range 100e900 m/z, scan
time 1 s) and a single ion recording mode (SIR dwell time 0.2 s) with
following parameters: capillary voltage 3.0 kV; cone voltage 35 V; extractor
and RF lens voltages were 2.0 and 0.2 V, respectively. Source and desolvation
temperatures were 120 C and 380 C, respectively, with the N 2 gas flow of 400
l\h. Chromatograms were recorded at specific m/z for each compound and,
due to the lack of specific standards for fla-vonoids, values are presented as
peak area normalization obtained by division of the HPLC peak area for each
compound with the lowest detected peak area. Quantification of 3-O-
caffeoylquinic acid (3-CQA) was made by external standard method, while
the quantities of other chlorogenic acids are presented as 3-CQA equivalent
amounts (mg 3CQAE/100 g). 3-CQA was obtained from Sigma-Aldrich
(Steinheim, Germany). Standard solutions were made by dissolving pure
compounds in methanol and further di-lutions in mobile phase. The data
acquisition and spectral evalua-tion for peak confirmation were carried out by
the Waters Empower 2 Software (Waters, Milford, USA).
2.2.9. Analysis of chlorophylls
Chlorophylls (a and b) were extracted from 100 mg ground corn silk and
medicinal herb samples with acetone 80% in a proportion of 1:10 (w/v), and
the absorbance of the supernatants were measured at 663 and 645 nm. The
content of chlorophyll a and chlorophyll b was calculated according to the
equations given by Wellburn (1994), and expressed as mg per 100 g of d.m.
2.2.10. Analysis of mineral matters
The ion chromatographic (IC) method was performed for determination of
þ þ 2þ 2þ
Na , K , Mg and Ca in corn silk samples. Wet micro mineralization was
applied for sample preparation using PTFE pressure digestion vessels. Briefly,
200 mg of powdered sample was digested with 2 mL of concentrated HNO 3.
The vessels were sealed and heated for 6 h in an oven at 150 C. The rest of
digestion was diluted with mineral-free water up to 25 ml. Before being
injected into the chromatograph, aliquots from the solutions were filtered
through a 0.22 mm membrane filter. The analysis was conducted using an ion
chromatograph Dionex ICS 5000 þ DP (Thermo Scientific, Waltham, MA,
USA) equipped with a conduc-tivity detector. The cations were separated
using Dionex Ion Pac™ CS12A - 5 mm (3 mm 150 mm) cation-exchange
column with 20 mM methanesulfonic acid as a mobile phase at a flow rate of
0.5 ml/min. Working standard solutions were prepared by dilution of Fluka
1000 mg/l solutions. Identified cation peaks were confirmed and quantified by
data acquisition and spectral evalua-tion using the Thermo Scientific Dionex
Chromeleon 7.0.
365
þ þ 2þ
Chromatography Data System Software. The content of Na , K , Mg and
2þ
Ca is expressed as mg per 100 g of d.m.
2.2.11. Measurement of pH
Grinded samples (0.4 g) were mixed with 20 ml of deionized water and
vortexed for 3 min. Mixtures were held at ambient temperature for 1 h to
separate solid and liquid phases. After centrifugation the pH of supernatants
was measured using a pH meter (MeterLab PHM210, France).
2.3. Statistical analysis
The analytical data are reported as mean ± standard deviation of at the
least three independent extractions. The results were sta-tistically analyzed
using Statistica software version 5.0 (StatSoft Co., Tulsa, OK, USA).
Significance of differences between samples was analyzed by the Tukey
(HSD) test. Differences with p < 0.05 were considered significant.
3. Result and discussion
3.1. Content of total phenolic compounds in corn silks and
medicinal herbs
Silk of different corn genotypes varies greatly in composition and content
of its phenolic compounds. In this study, the content of total phenolics,
flavonoids, anthocyanins and proanthocyanidins of purple, green, pinky and
yellow corn silk is shown in Table 1. The study reports a significantly higher
content of total phenolic and flavonoid compounds in corn silk at the silking
stage than it was reported by Sarepoua et al. (2015). The contents of 10160.8
mg CGAE/100 g and 6478.3 mg CE/100 g, respectively, were highest in the
silk of the hibryd ZP 341. According to our results, flavonoids were the most
common class of phenolics in the silk. Anthocyanins, as subgroups of
flavonoids, were present only in silk of ZP 341 and ZP Exp hybrids. The
contents of anthocyanins of 1.57 and 192.96 mg CGE/100 g contributed to
their pinky and purple color of silk, respectively. Although Maksimovic et al.
(2005) measured proan-thocyanidins in silk of all investigated genotypes
grown in Serbia, our results indicated the presence of proanthocyanidins only
in purple colored silk. In traditional healthcare, silk is mainly used from corn
at the R4 to R6 stage when it takes brown color. However, this study was
carried out to determine phenolic compounds content in both, fresh and
mature-brown corn silk, as well as to determine the maturity stage in which
corn silk can be most effi-ciently used as tea or food ingredient rich in
bioactive compounds. According to our results, corn silks at the silking stage
are much more suitable for use as a source of phenolic compounds than silks
at the R4 dough stage. The content of total phenolic and flavonoid compounds
in fresh silks were higher by about 2- to 4-fold in relation to that in silks at the
R4 dough stage. After 25 days of emergence, silk of the hibryd ZP 555 had
the lowest content of analyzed bioactive compounds (2093.9 mg CGAE/100 g
and 1840.1 mg CE/100 g, respectively). As a consequence of chemical
changes during aging, anthocyanins and proanthocyanidins were detected in
trace amounts in silk of ZP Exp hybrid (Table 1). Namely, the polyphenols are
stable as long as they are accumulated in living plant cells. When tissues
undergo physiological changes, such as corn silk aging, some of the
polyphenols are chemically converted to secondary polyphenols by enzymatic
and non-enzymatic re-actions (Tanaka et al., 2010). During silk aging,
phenolic compounds undergo enzymatic oxidation resulting in the production
of qui-nones which condense with themselves or proteins to produce brown-
stated that black tea
colored complexes. Tanaka et al. (2010)
polyphenols are a typical and economically important
example of
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S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370

Table 1
Total phenolic compounds content in corn silks and medicinal herbs.

Samples Total phenolics (mg CGAE/100 g) Total flavonoids (mg CE/100 g) Anthocyanins (mg CGE/100 g) Proanthocyanidins (mg LE/100 g)
Corn silk-5 day after emergence 73.5
cd
± 40.9
c
192.9 ± 0.3
a
69.4 ± 6.6
c

ZP Exp 8101.6 ± 5565.3

ZP 555 8382.4 ± 147.1cd 5608.7 ± 307.4bc n.d. n.d.
ZP 341 10160.8 ± 250.0b 6478.3 ± 409.9a 1.57 ± 0.1
b
n.d.
ZP 366 8372.0 ± 308.9cd 5514.5 ± 92.2c n.d. n.d.
Corn silk-25 day after emergence
ZP Exp 3958.9 ± 378.5g 3594.2 ± 143.5
d
1.49 ± 0.1
b
0.25 ± 0.01
d

ZP 555 2093.9 ± 215.7h 1840.1 ± 40.9

f
n.d. n.d.
ZP 341 4347.2 ± 49.0fg 3644.9 ± 297.2
d
n.d. n.d.
ZP 366 3674.7 ± 333.4g 2985.5 ± 20.5
de n.d. n.d.
Medicinal herbs 397.1
ef
± 143.5
e

Mentha piperita 5647.2 ± 2681.2 n.d. n.d.

ab
Melissa officinalis 11315.2 ± 764.8b 6420.3 ± 225.5 n.d. n.d.
ef
Ginkgo biloba 6281.6 ± 588.3e 2420.3 ± 204.9 n.d. 3251.4 ± 11.7
a
d
Thymus serpyllum 8636.0 ± 29.4c 3608.7 ± 245.9 n.d. n.d.
Green tea 15423.2 ± 367.7a 6652.2 ± 143.5
a
n.d. 1480.7 ± 10.4
b

Salvia officinalis 6999.2 ± 44.1de 3594.2 ± 81.9

d n.d. n.d.

Mean of samples followed by the same letter within the same column are not significantly different (p > 0.05). n.d.- not detected.

secondary polyphenols. In contrast, the polyphenol composition of
commercial green tea and other herbs that undergo minimal oxidation during
processing is similar to that of fresh tea leaves due to enzymes inactivation.
The commercial green tea had the higher content of total phenolic and
flavonoid compounds (15423.2 mg CGAE/100 g and 6652.2 mg CE/100 g,
respectively) in comparison with corn silks at the silking and R4 dough
stages. However, corn silks at the silking stage were characterized by higher
content of total phenolic compounds, in particular a flavonoids, than Mentha
piperira, Ginkgo biloba, Thymus serpyllum and Salvia officinalis. The
presence of anthocyanins was not detected in medicinal herbs. However,
compared to the corn silk, a high content of proantho-cyanidins was measured
in samples of Ginkgo biloba and green tea (3251.39 and 1480.75 mg LE/100
g, respectively). According to our research, the contributions of flavonoids to
the content of total phenolic compounds of medicinal herbs were significantly
lower (38.53e56.73%) than that in corn silks at silking, as well as the R4
dough stage (63.75e68.69% and 81.24e90.80%, respectively). As active
ingredient in corn silk, the flavonoids possess various phar-macological
activities and may relate to silk beneficial effects on human health.
standard, while the 3-CQA equivalent amount was given for 4-CQA, 5-CQA
and p-CQA (Table 2). According to our study, corn silks at the silking stage
were a rich source of chlorogenic acids. 3-CQA and 4-CQA exhibited a broad
range of variation from 932.7 to 1840.8 mg/ 100 g and 186.9e426.6 mg 3-
CQAE/100 g, respectively, in silks at the silking stage. In comparison to
yellow silk of the hybrid ZP 366, purple and green silk of ZP Exp and ZP 555
hybrids had a higher content of 3-CQA and 4-CQA by about 2-fold. The
content of 5-CQA and p-CQA was less than 100 mg 3-CQAE/100 g in all
silks at the
silking stage. According to Hadzi-Taskovic Sukalovic et al. (2010), the
content of 3-CQA in unpollinated corn silks was significantly lower. By
comparison, the highest sources of chlorogenic acid is considered to be coffee
having a content of 3-CQA from 478.2 to 924.8 mg/100 g (Farah et al., 2005).
As a consequence of polyphenol oxidase activities the chlorogenic acids were
oxidized and detected in corn silks after 25e27 days of their emergence in low
concen-tration (Table 2). The breakdown of chlorogenic acids is very
important to the flavor and color of corn silk tea. The content of 3-CQA in silk
at the R4 dough stage ranged from 22.6 to 49.9 mg/100 g that was lower by
96e98% in relation to that in fresh silk. In contrast, 5-CQA and p-CQA were
more stable.

3.2. Phenolic acid profiles of corn silks

Five chlorogenic acids (CGA) were detected in the corn silk samples by
the LC/MS analysis (Fig. 1). More precisely, these are the esters of
hydroxycinnamic acids, caffeic or p-coumaric, and quinic acid under the
common name of chlorogenic acids. The quinic acid increase is driving factor
in acidity development (Balzer, 2001). pH determination of water extracts
showed the pH value of corn silks at the silking and R4 dough stages of
5.23e5.73. 3-CQA was major chlorogenic acid found in corn silks followed
by 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA) and p-
coumar-oylquinic acid (p-CQA). However, little has been published on the
hydroxycinnamic acids esters from corn silk. Chlorogenic acids have a strong
antioxidant properties. Furthermore, Xu et al. (2012) reported that three
caffeoylquinic acid isomers showed quite similar antioxidant activities,
indicating that the position of ester-ification on the quinic moiety of
caffeoylquinic acid had no effect on its antioxidant activities. Generally,
chlorogenic acids exhibit many biological properties and have been associated
with a series of health benefits. In this study, 3-CQA was quantified using
external
3.3. Flavonoid profiles of corn silks
Corn silk is a rich source of phenolic compounds, especially flavonoids.
According to thin-layer chromatography results, fla-vonoids from corn silk
were mainly composed of luteolin, for-mononetin and apigenin, while the
results of ultraviolet scanning showed that the flavonoids of corn silk were
mainly flavones and isoflavones (Yu et al., 2008). Our results are in well
accordance with these data, as well as those reported by Ren et al. (2009).
Three flavone glycosides of corn silk were identified by LC/MS analyses.
Luteolin derivates such as maysin [rhamnosyl-6-C-(4-ketofucosyl)-5,7,30,40-
tetrahydroxyflavone] and derivative of its methoxy analogue was dominant
(Fig. 1). Most of the flavone glycosides showed a strong scavenging activity
against radicals and may be beneficial natural food antioxidants. Through its
antioxidative action, maysin as the most abundant corn silk flavonoid had
neuroprotective effects against oxidative stress (Choi et al., 2014) and a
strong therapeutic potential for the treatment of cancer (Yang et al., 2014). In
this study, normalized amounts are given for identified dominant flavonoids
(Table 2).
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S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370

Fig. 1. LC-MS (A) and UV (B) chromatograms of corn silk methanolic extract. Peaks are noted as follows: 1. 5-O-Caffeoylquinic acid (353 m/z); 2. 3-O-Caffeoylquinic acid (353 m/z);
3. 4-O-Caffeoylquinic acid (353 m/z); 4. Caffeoylquinic acid derivative (353 m/z); 5. p-Coumaroylquinic acid (337 m/z); 6. Maysin derivative (593 m/z); 7. Maysin (575 m/z); 8. Methoxymaysin
derivative (607 m/z).

Table 2
Content of identified chlorogenic acids and flavonoids in corn silks.

5-O-Caffeoylquinic acid 3-O-Caffeoylquinic acid 4-O-Caffeoylquinic acid p-Coumaroylquinic acid Maysin Maysin** Methoxymaysin
(353 m/z) (353 m/z) (mg/100 g) (353 m/z) (337 m/z) derivative** (579 m/z) derivative**
(mg 3CQAE/100 g)* (mg 3CQAE/100 g)* (mg 3CQAE/100 g)* (593 m/z) (607 m/z)
Corn silk-5 day after emergence
ZP Exp 86.5 ± 2.8a 1840.8 ± 3.1a 362.1 ± 11.5b 44.7 ± 0.9
c
39.5 ± 2.8a 122.7 ± 0.5a 66.6 ± 5.6
a

ZP 555 81.3 ± 1.8

ab
1816.6 ± 7.6a 426.6 ± 12.6a 90.9 ± 2.9
a
38.8 ± 3.5a 120.2 ± 6.3a 63.1 ± 1.6
a

ZP 341 88.9 ± 3.9

ab
1408.2 ± 16.3b 240.1 ± 14.9c 43.4 ± 1.6
c
28.9 ± 2.5b 93.9 ± 6.3b 45.6 ± 3.7
b
b 932.7 ± 4.4c 186.9 ± 8.1d 73.5 b 25.9 ± 2.3b 83.9 ± 5.3c 41.4 b
ZP 366 74.4 ± 1.6 ± 2.0 ± 1.7
Corn silk-25 day after emergence
ZP Exp 52.7 ± 1.9c 30.4 ± 3.1d 29.3 ± 1.6e 36.2 ± 1.0
de
2.9 ± 0.3c 12.6 ± 1.2de 4.6 ± 0.2
c
e
ZP 555 26.8 ± 1.1 22.6 ± 0.9d 21.2 ± 0.5e 31.1 ± 0.9
e
1.1 ± 0.1c 4.7 ± 0.4f 1.2 ± 0.1
c

ZP 341 39.9 ± 2.3d 49.9 ± 1.4d 25.2 ± 1.8e 31.8 ± 1.1

e
3.6 ± 0.4c 17.1 ± 1.7d 5.0 ± 0.1
c

ZP 366 30.6 ± 2.6de 34.4 ± 1.6d 22.1 ± 0.6e 38.6 ± 2.0

cd 3.4 ± 0.3c 14.5 ± 1.2d 4.9 ± 0.3
c

Mean of samples followed by the same letter within the same column are not signi ficantly different (p > 0.05); *amount of chlorogenic acids expressed as mg of 3-O-caf-feoylquinic acid equivalent
(3CQAE) per 100 g of d.m.; **normalized amounts are given for identified flavonoids.

The silks of ZP Exp and ZP 555 hybrids at the silking stage had content of
maysin (575 m/z), another maysin derivative (593 m/z) and methoxymaysin
derivative (607 m/z) higher by about 1.5-fold than silk of the hybrid ZP 366.
These results indicated a high positive correlation between the content of
luteolin derivatives and chlorogenic acid (3-CQA) in silks. As a consequence
of silk aging and flavonoids oxidation, after 25e27 days of emergence the
contents of maysin, maysin derivative and methoxymaysin de-rivative in
mature-brown corn silks were lower by about 6- to 25-fold, 8- to 35-fold and
8- to 52-fold, respectively, in relation to that in fresh silks. By oxidation
incurred quinoidal compounds contributed to the formation of heterogeneous
polymers respon-sible for browning reaction and silk color.
3.4. Content of chlorophylls in corn silks and medicinal herbs
There are few papers dealing with the non-polyphenolic con-stituents of
corn silk, e.g. other plant pigments such as chlorophylls and carotenoids. In
this study, contents of chlorophyll a and b in investigated corn silk and herbs
are presented in Table 3. With the
exception of silk of ZP Exp hybrid at the silking stage, chlorophyll a was
higher by about 55e63% and 10e43% than chlorophyll b in all other corn silks
at the silking and R4 dough stages, respectively. Compared with medicinal
herbs, the content of total chlorophyll in corn silks was low. Mentha piperita
had the highest level of chlo-rophyll a and b (774.13 and 356.75 mg/100 g,
respectively), while silk of the hybrid ZP 366 at the R4 dough stage had the
lowest content (4.42 and 3.99 mg/100 g, respectively). Our results indicate
that intra-species variation in chlorophyll a and b levels was highly
statistically significant, while within corn species the variations were seem to
occur depending on the maturity stage. Chlorophyll, responsible for the
characteristic green color, can degrade to un-desirable grey-brown compounds
such as pheophorbide and pheophytin (Heaton and Marangoni, 1996) during
silk senescence. As a consequence of aging, after 25 days of emergence the
contents of chlorophyll a and b in mature-brown corn silks on average were
lower by about 4.8-fold in relation to that in fresh silks. According to the
results of Higashi-Okai et al. (2001),
chlorophyll a and its oxidized
derivative pheophytin a had a higher antioxidant activity than
some carotenoids. The ranks of suppressive activity against
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S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370

Table 3 activity being the highest in silk of the hybrid ZP 341 at silking stage. This
Content of chlorophyll in corn silks and medicinal herbs. activity was higher by about 71% than that found in the silk at the R4 dough
Samples Chlorophyll a (mg/100 g) Chlorophyll b (mg/100 g) stage. Although the purple silk of ZP Exp hybrid had a lower content of total
Corn silk-5 day after emergence phenolic compounds, its high antiox-idant activity at the silking stage could
ZP Exp 29.6 ± 0.5g 54.0 ± 1.3ef be related to the specific composition of anthocyanin and hydroxycinnamic
ZP 555 57.9 ± 0.9f 22.4 ± 1.8fg acids esters.
ZP 341 55.5 ± 0.5f 22.9 ± 0.2fg
ZP 366 55.3 ± 0.5f 24.6 ± 0.2fg
Investigations of Zilic et al. (2012) also demonstrated that pig-mented and
Corn silk-25 day after emergence
anthocyanin-rich corn kernels showed a higher radical scavenging activity
ZP Exp 12.3 ± 1.3gh 7.0 ± 0.3g than non-pigmented samples. In addition, the content of 3-CQA and maysin
ZP 555 12.4 ± 1.4gh 7.2 ± 0.3g 2
was highly positively correlated with the antioxidant activity of corn silks (r
ZP 341 12.2 ± 1.2gh 7.6 ± 0.1g 2
ZP 366 4.4 ± 0.1h 4.0 ± 0.4g ¼ 0.88 and r ¼ 0.91, respectively). On average 82% lower antioxidant
Medicinal herbs ± 15.7
a
± 31.2
a activity of silks at the R4 dough stage in comparison to that of fresh samples
was the result of polyphenols oxidation and their derivates formation dur-ing
Mentha piperita 774.1 356.8 silk maturation and aging. Despite that, the antioxidant activity of mature-
Melissa officinalis 492.9 ± 9.1b 239.6 ± 10.8b brown silk was still high. Fifty percents of the DPPH radical was inhibited by
Ginkgo biloba 334.9 ± 1.7c 149.5 ± 11.9c 0.151e0.239 mg of silk. By comparison, the amount of wholemeal from 1.38
Thymus serpyllum 147.5 ± 5.8e 68.5 ± 3.5de
to 10.69 mg of different cereals
Green tea 194.8 ± 2.2d 54.4 ± 3.4ef
Salvia officinalis 213.2 ± 5.7d 102.1 ± 8.8d
was needed to scavenge 50% of the DPPH radical (Zilic et al., 2011).
Mean of samples followed by the same letter within the same column are not signi ficantly
According to our study, the antioxidant activity of six investigated herbs was
different (p > 0.05).
high with IC50 values ranging from 0.023 to 0.085 mg. Green tea and Mentha
piperita had the highest and lowest antiox-idant activity, respectively. The
hydroperoxide generation were chlorophyll a > lutein > pheophytin a > antioxidant activity of corn silks at the silking stage can be compared with
chlorophyll b > b-carotene > pheophytin b. that of medicinal herbs such as Melissa officinalis, Ginkgo biloba, Thymus
serpyllum and Salvia officinalis. However, compared with green tea,
anthocyanin-rich corn silk of ZP Exp and ZP 341 hybrids had a lower
3.5. Antioxidant activity of corn silks and medicinal herbs antioxidant activity by 2.0-fold, while it was lower by 3.2-fold in green and
yellow silk of ZP 555 and ZP 366 hybrids. On the other hand, the IC50 values
As it is shown in this study, corn silk is an important source of natural
of purple and pinky corn silk at the silking stage were similar to that of
bioactive compounds with strong antioxidant properties. The IC50 values of Melissa officinalis and higher by about 1.8-fold than that of Mentha piperita
DPPH radical scavenging activity of corn silk samples evaluated across the and Salvia officinalis. If the DPPH radical scavenging activity is considered,
two maturity stages ranged from 0.043 to 0.239 mg of d.m. Corn silks at the corn silk should be harvested at the silking stage.
silking stage had the highest antioxidant activity with IC 50 value of 0.059 mg
(on average) followed by silks at the R4 dough stage (0.180 mg, on average)
(Fig. 2). Obtained results are well in accordance with data reported by
Sarepoua et al. (2015). According to the study carried out by Ebrahimzadeh et
al. (2008) the percentage of DPPH radical scavenged by corn silk ethanolic 3.6. Mineral matters in corn silks
extract was 92.6 at the concen-tration of 1.6 mg/ml. As expected, a higher
content of total phenolic compounds in corn silks contributed to their higher Mineral content (K, Na, Mg, Ca) of corn silks at the silking and R4 dough
antioxidant stages is shown in Table 4. In general, the content of the major elements
varied considerably among silk samples. Fresh and

Fig. 2. DPPH scavenging activity of corn silks and medicinal herbs. MP-Mentha piperita, MO-Melissa officinalis, GB-Ginkgo biloba, TS-Thymus serpyllum, GT-Green tea, SO-Salvia officinalis.

Bars with different letters are statistically significantly different (p < 0.05).
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S. Zilic et al. / Journal of Cereal Science 69 (2016) 363e370
Table 4

Content of mineral matters in corn silks.

Corn samples Na (mg/100 g) K (mg/100 g) Mg (mg/100 g) Ca (mg/100 g)

Corn silk-5 day after emergence
f bc
ZP Exp 10.1 ± 1.1 1656.2 ± 89.4 88.6 ± 8.9c 63.2 ± 1.1cd
e ab
ZP 555 13.9 ± 0.8 1717.0 ± 40.2 86.8 ± 2.8c 59.5 ± 0.6cd
b c
ZP 341 25.6 ± 0.2 1550.7 ± 50.2 90.0 ± 4.4c 53.9 ± 0.8d
ZP 366 18.3 d 1693.9 ab 97.2 ± 5.2bc 65.4 ± 5.4c
± 0.8 ± 46.7
Corn silk-25 day after emergence
e c
ZP Exp 12.5 ± 0.5 1520.9 ± 26.2 125.6 ± 0.7a 133.9 ± 2.8a
c d
ZP 555 21.9 ± 0.6 1359.9 ± 46.6 130.4 ± 4.5a 137.3 ± 6.0a
a d
ZP 341 28.9 ± 0.3 1334.1 ± 38.0 104.4 ± 2.1b 121.8 ± 3.5b
ZP 366 18.4 d 1832.6 a 135.3 ± 2.3a 139.9 ± 2.1a
± 0.3 ± 37.5
Mean of samples followed by the same letter within the same column are not significantly different (p > 0.05).

mature-brown silk of the hybrid ZP 341 had the highest Na content (25.61 and
28.93 mg/100 g, respectively) and the lowest K content (1550.7 and 1334.1
mg/100 g, respectively). On the contrary, there was a high positive correlation
between Mg and Ca contents. The highest content of both was found in silk of
the hybrid ZP 366. It can be observed that the mineral content depends on silk
maturity stage. As for Na, Mg and Ca content, the fresh silks exhibited lower
values compared to the silks at the R4 dough stage, and vice versa as far as K
content. After 25 days of emergence, on average the content of Na, Mg and
Ca was increased by about 17, 27 and 55%, respec-tively. Although 15 and
20% of Mg was associated with chlorophyll (White and Broadley, 2009), the
degradation of chlorophyll was not accompanied by a reduction of the Mg
content. According to Rahman and Wan Rosli (2014), immature silk was a
richer source of Ca, Mg, Cu and Zn than mature silk, while other minerals
such as K, Na, Fe and Mn were found to be in a higher amount in mature silk.
Generally, silk is a very rich source of K. In our study, the content of K in
silks at silking stage ranged from 1550.7 to 1717.0 mg/100 g (Table 4). On
average, the content of Na, Mg and Ca was lower by about 97-, 18- and 27-
fold, respectively, than the K content in fresh corn silks. Decreasing order: K
> Mg > Ca > Na is in accordance with data reported by Kim et al. (2000).
According to results of Velazquezd et al. (2005) the diuretic effect of corn silk
was attrib-uted to its high K content. In addition to this effect, which was
accompanied by increased excretion of Na and K from the body, K has been
reported to attenuate vascular contractions and to reduce the elevated blood
pressure. Humans require large amounts of K, Mg and Ca in their diet, and K
is generally consumed in adequate amounts. Given the Mg and Ca
deficiencies are common in many developed and developing countries, our
results indicate that silks at the R4 dough stage are much more suitable for
use as a source of these micro- and macrominerals than silks at silking stage.
confirms the high level of potassium in corn silk which is asso-ciated with its
diuretic effect. In contrast to the fresh silk, silk at the R4 dough stage is much
more suitable for use as a source of micro- and macrominerals.
Acknowledgements
This work was financially by the Ministry of Education, Science and
Technological Divelopment of the Republic of Serbia (Grants no. TR 31069
and OI 173040).
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