Phenolic Compounds and Antioxidant Activities of Corn Silk (Zea mays L.) Extract and
Its Application in Food Products
Samonwan Boonchan
1,
*, Aikkarach Kettawan
2
, Uthaiwan Suttisansanee
2
, Anadi
Nitithamyong
2,
#
1 Food Science for Nutrition Program, Institute of Nutrition, Mahidol University, Thailand
2
Institute of Nutrition, Mahidol University, Thailand
*e-mail: b.samonwan@gmail.com, #e-mail: anadi.nit@mahidol.ac.th
Abstract
Corn silk ( Zea mays L .) is one of the industrial waste products from corn milk processing in
Thailand. The purpose of this study was to prepare an extract of corn silk and determine its phenolic content, antioxidant activities and potential application in foods. Fresh corn silk was extracted at varying temperatures (40, 50, 60 and 70 °C) using 95% ethanol as a solvent.
Then the phenolic content of the extract was quantified. Its antioxidant activities were screened using the 1,1-diphenyl-2-picryl hydrazyl (DPPH•) radical scavenging and oxygen radical absorbance capacity (ORAC). The results showed that extraction at 70 o
C yielded the highest content of phenolic compounds and total phenolics (9.71 ± 0.28 µg/ml sample and
2.02 ± 0.08 mg GAE/ml sample, respectively). The extract also exhibited the highest DPPH radical scavenging activity (5.81 ± 0.17 µmol TE/ml sample) and oxygen radical absorbance capacity (70.68 ± 11.14 µmol TE/ml sample). The predominant phenolics in corn silk extract included feluric and p-coumaric acids.
Keywords: corn silk, extraction, phenolic compounds, antioxidants, antioxidant activities
Introduction
Corn silk ( Zea mays L.
) is a part of the by-product from the corn manufacturing industries in Thailand. These wastes or by-products are normally discarded or transformed to animal feeds. Nevertheless, corn silk still contains beneficial substances such as the flavonol glycosides of quercetin, isorhamnetin and kaempferol [1] and lipids [2]. It is rich in phenolic compounds known to significantly affect human health, such as anthocyanins, p-coumaric acid, vanillic acid, protocatechuic acid, derivatives of hesperidin and quercetin, and bound hydroxycinnamic acid forms composed of p -coumaric and ferulic acid [3]. Phenolics are antioxidants with redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. Phenolic compounds exhibit a wide range of physiological properties, such as antiallergenic, anti-artherogenic, anti-inflammatory, antimicrobial, antioxidant, antithrombotic and cardio-protective effects [4]. However, literature revealed that limited information exists about the utilization in foods of corn silk as a source of phenolic compounds and the work studying its antioxidant properties. Therefore, the objectives of this study were to prepare an extract of corn silk, determine the phenolic compounds and antioxidant activities and investigate its potential application as an antioxidant by incorporating into foods.
Methodology
1. Sample and chemicals
Corn silk used for this investigation was obtained from a corn milk processing facility, the National Corn and Sorghum Research Center (Suwan Farm), Nakhon Ratchasima

Province. All chemicals used for the determination of total phenolic contents and antoxidant activities, such as DPPH (1,1-diphenyl-2-picryl hydrazyl), Folin-Ciocalteu reagent, Trolox
((±)-6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid), APPH (2'-azobis(2amidinopropane)dihydrochloride), Fluorescein and Gallic acid monohydrate were analyical grade, and were purchased from Sigma-Aldrich, Germany. Ethyl acetate, acetonitrile and methanol (HPLC grade) used for determination of total flavonoid contents were purchased from RCI Labscan, Thailand. Ethanol used as a solvent for extraction of sample was also of analytical grade, and was purchased from Merck, USA.
2. Sample preparation
After collection, the corn silk sample was cleaned by removing the dry parts and dirty particles, packaged in polyethylene bags and stored at -20 o
C until used in further experiment.
3. Sample extraction
The corn silk sample was milled in a high speed mill (Moulinex Optiblend Duo,
KRUPS, France) and the prepared sample was extracted by a modified alcoholic method of
Liu et al . [5]. The sample was extracted in a water bath shaker with 95% ethanol (1:4 g/ml) using extraction time of 24 h by varying the extraction temperature (40, 50, 60, and 70 o
C).
The extract was then filtered through a Whatman No.1 filter paper to remove the residue of corn silk, condensed by a rotary evaporator (Buchi R-114 Rotary Evaporator, Switzerland) to remove the solvent. Finally, the corn silk extract was adjusted volume to 25 ml by deionized water and stored at 4 o
C for subsequent analyses.
4. Determination of phenolic acids by HPLC
Phenolic acids in corn silk extract were determined by a modified alkaline hydrolysis method of Tian et al . [6] and Beta et al.
[7]. Three ml of extract were hydrolyzed with 30 ml of 4N NaOH. The head-space of the Erlenmeyer flask was flushed with a stream of N
2
to remove the air and the flask was shaken at room temperature for 4 h in the absence of light.
Samples were then acidified with 6N HCL to pH 1.0-1.5 and extracted with ethyl acetate
(twice the volume, two times). The supernatant was condensed by a rotary evaporator to remove the solvent, then the dried alkaline extracts were re-dissolved in 3 ml of MeOH/H
2
O
(50/50, v/v) and sonicated for 5 min. Extracts were filtered with a 0.22 µm PTFE filter and measured using an HPLC system with a photodiode array detector set at a wavelengths of
190-150 nm. Phenolic compounds in the samples were identified by comparing their relative retention times and UV-Vis spectral data to known, previously injected standards.
5. Determination of total phenolic content
The total phenolic content was analyzed by Folin–Ciocalteu method of Lu et al . [8] and Phomkong et al . [9] with a modification in the technique of measurement using a spectrophotometer. The extract was diluted with 70% ethanol (v/v) in a proper dilution. Then
0.5 ml of the extract or standard solution of gallic acid (10, 20, 40, 60 and 80 µg/ml) was added in a tube containing 2.25 ml distilled deionized water. After that 0.25 ml of Folin–
Ciocalteu reagent was added to the mixture and shaken. The mixture was left to stand for 5 min, then 2 ml of 7.5% Na
2
CO
3
solution (w/v) were added and the mixture incubated at room temperature for 2 h. For the blank sample, Folin–Ciocalteu reagent in the mixture was substituted with the same volume of distilled deionized water. Absorbance of each reaction mixture was measured at 760 nm using a UV-Vis spectrophotometer. The results were expressed as milligrams of gallic acid equivalent (mg GAE) per ml of sample.

6. Determination of antioxidant activities
6.1. DPPH radical scavenging activity
The DPPH radical scavenging activity was determined according to the method described by Binsan et al . [10] with some modification. The extract was diluted with 95% ethanol in a proper dilution and 1 ml of sample extract was added with 2 ml of DPPH in 95% ethanol. The reaction mixture was shaken vigorously and allowed to stand for 30 min at room temperature, under dark condition. The absorbance of each reaction mixture was measured at
517 nm by a UV-Vis spectrophotometer. The percentage of DPPH radical scavenging activity
(%) of the sample was calculated as follows:
% Radical scavenging activity (RSA) = (Blank absorbance – Sample absorbance) x 100
Blank absorbance
6.2. Oxygen radical absorbance capacity (ORAC)
The ORAC assay was carried out by the method of Huang et al . [11]. The extract was diluted with a working phosphate buffer solution pH 7.2 in a proper dilution and 500 µl of diluted sample or Trolox standard or blank (buffer solution) and 3 ml of warmed (37 o C) fluorescein working solution were added into a cuvette with continuously mixing using a magnetic stirrer. Finally, AAPH solution was added. The intensity of fluorescence was monitored by a spectrofluorometer with an excitation wavelength of 493 nm and an emission wavelength of 515 nm. The area under the fluorescence decay curve (AUC) and ORAC values were calculated as follows:
AUC = (0.5 + ƒ5/ ƒ4 + ƒ/ ƒ4 + ƒ7/ ƒ4 + … + ƒi/ ƒ4) x CT
ORAC value = ((AUCsample - AUCblank /(AUCTrolox – AUCblank)) x dilution factor
Where ƒ4 is initial fluorescence reading at cycle 4, ƒi is fluorescence reading at cycle
I and CT is cycle time in minutes. The results were expressed as micromoles of Trolox equivalent (µM TE) per ml of sample.
7. Statistical analysis
Results are expressed as mean ± standard deviation (SD) of triplicate determinations.
The statistical analyses were conducted using SPSS software. One way analysis of variance
(ANOVA) and Duncan’s multiple-range test were used to determine the differences among the means. P values of < 0.05 were regarded as significant.
Results
1. Quantification of phenolic acids by HPLC
Phenolic acids of corn silk extracted at different extraction temperatures are given in
Table 1. The dominant phenolic acids in the extracts were ferulic acid and p -coumaric acid.
There was a significant difference among the phenolic acid content of extracts from all extraction temperatures ( p <0.05) with the values being 9.71 ± 0.28, 8.49 ± 0.27, 7.56 ± 0.24 and 6.62 ± 0.15 µg/ml sample, for the extract from extraction at 70 o
C, 60 o
C, 50 o
C and 40 o
C, respectively.
2. Total phenolic contents in corn silk extracts
Total phenolic contents shown in Table 2 reveal a significant difference between total phenolic content of the extracts from all extraction temperatures ( p <0.05). The phenolic content of the extract from extraction at 70 o
C presented the highest level among the four extracts, 2.02 ± 0.08 mg GAE/ml sample, followed by the extract from 60 o
C with a value of
1.83 ± 0.07 mg GAE/ml sample, the extract from 50 o
C with a value of 1.62 ± 0.03 and the

extract from 40 o
C showed the lowest content (1.43 ± 0.19 mg GAE/ml sample).
3. Antioxidant activity of the extracts
Antioxidant activities of corn silk extracts were determined by using DPPH and
ORAC methods and the results are presented in Table 3. DPPH value of corn silk extracts was significantly different among all the extracts from varying extraction temperatures
( p <0.05). The DPPH values of all the extracts were found to be 5.81 ± 0.17, 4.72 ± 0.15, 3.97
± 0.16 and 3.53 ± 0.09 µmol TE/ml sample, for 70 o
C, 60 o
C, 50 o
C and 40 o
C, respectively. In term of ORAC value, there was a significant difference of extraction at 70 o
C (p<0.05); but there was no difference between the remaining three extraction temperatures (40 o
C, 50 o
C and
60 o C). A significant correlation was found between the total phenolic contents and the DPPH value (R
2
= 0. 9664) (Fig.1a) and ORAC value (R
2
= 0.7063) (Fig.1b).
Discussion and Conclusion
The corn silk was extracted with 95% ethanol at the temperatures of 40, 50, 60 and
70 o
C and extraction times of 24 h. The contents of phenolic compounds increased with increasing of extraction temperature, particularly when the temperature was raised to 70 o C.
This is probably due to the fact that this temperature is near the boiling point of the solvent
(79 o
C), at which point the bubbles were formed and their random motion caused greater mixing for the system. The effects could be explained by the increase in solubility and the speed of the molecular movements at higher temperature, which caused the extracting agent to diffuse more quickly into the corn silk sample, and caused the phenolic compounds to diffuse more quickly from the corn silk sample to the extracting agent [12]. To confirm the major antioxidant capacity contributors in corn silk extracts, the antioxidant activity (AOA) evaluated by DPPH assay and ORAC assay was correlated with the content of total phenolics
(TPC). Fig. 1 demonstrated that the content of total phenolics agreed with antioxidant capacity assays. An excellent correlation (R
2
=0.9664) between DPPH and TPC can be explained by the similar mechanism occurring in both assays; electron transfer reaction [13].
A less strong correlation (R
2
=0.7063) was found between ORAC and TPC.
In summary, this study was designed to evaluate the contents of phenolic and antioxidant activities of extract from corn silk. The content of phenolic compounds for extract from extraction temperature at 70 o C showed better antioxidant capacities than other extraction temperatures in terms of TPC, DPPH radical scavenging activity, and oxygen radical absorbance capacity (ORAC). Correlation analyses suggested that phenolic acids were the main compounds responsible for the antioxidant potential of corn silk extracts. The abundant phenolic acids found in extracts were feluric acid and p-coumaric acid and the present investigation may be considered for the utilization of corn silk as a beneficial substances as well as the possibility of using corn silk a source of low cost natural antioxidant.

2,5 (a)
2
1,5 y = 0,2478x + 0,6055
R² = 0,9339
1
0,5
0
0 1 2 3 4
DPPH (µmol TE/ml sample)
5 6 7
2,5 (b)
2 y = 0,0121x + 1,0891
R² = 0,4988
1,5
1
0,5
0
0 20 40 60
ORAC (µmol TE/ml sample)
80 100
Figure 1.
Correlation of DPPH value with total phenolic content (TPC) (a); correlation of ORAC value with total phenolic content (TPC) (b).
Table 1.
Contents of detected phenolic acids in corn silk extracts.
T (ᵒC)
40
50
67
70
Phenolic acids (µg/ml sample)
p-COA
4.09 ± 0.10
d
4.67 ± 0.15
c
5.29 ± 0.15
b
5.80 ± 0.15
a p -COA, p -coumaric acid; FA feluric acid.
FA
2.53 ± 0.05
2.89 ± 0.08
d c
3.20 ± 0.11
3.91 ± 0.13
b a
In total
6.62 ± 0.15
d
7.56 ± 0.24
c
8.49 ± 0.27
b
9.71 ± 0.28
a
Value are mean ± standard deviation of triplicate analyses; different letters in same column indicate significant difference ( p <0.05). Results are ranked in ascending order; a>b>c>d.

Table 2. Total phenolic content (TPC) of corn silk extracts.
T (ᵒC)
40
50
67
70
TPC (mg GAE/ml sample)
1.43 ± 0.19
d
1.62 ± 0.03
c
1.83 ± 0.07
b
2.02 ± 0.08
a
Value are mean ± standard deviation of triplicate analyses; different letters in same column indicate significant difference ( p <0.05). Results are ranked in ascending order; a>b>c>d.
Table 3. Antioxidant activity of corn silk extracts.
40
50
67
70
T (ᵒC) Antioxidant activity (µmol TE/ml sample)
DPPH
3.53 ± 0.09
d
3.97 ± 0.16
c
4.72 ± 0.15
b
5.81 ± 0.17
a
ORAC
41.01 ± 11.53
47.47 ± 6.87
51.05 ± 0.17
b b
70.68 ± 11.14
b a
Value are mean ± standard deviation of triplicate analyses; different letters in same column indicate significant difference ( p <0.05). Results are ranked in ascending order; a>b>c>d.
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