ANTIOXIDANTS

HAN SUB KWAK1,2, SEOKGEUN JI1, MISOOK KIM1,2, YOUNGSEUNG LEE1,2, YOONHWA JEONG1,2*
*Corresponding author
1. Department of Food Science and Nutrition, Dankook University, Yongin-si, 448-701, Korea
2. Institute of Global Food Industry, Dankook University, Yongin-si, 448-701, Korea

Yoonhwa Jeong

Antioxidant activity

and total polyphenol content of coffee extracted

by three extraction methods
KEYWORDS: Coffee, extraction, antioxidant, polyphenol.

Abstract

Eight kinds of Arabica coffee beans were extracted using solid-liquid (SLE), espresso (ESE), and coffee
maker (CME) extractions. The antioxidant activity by DPPH assay and total polyphenol content (TPC)
were significantly lower in ESE compared to the other two extracts (p < 0.05). In FRAP assay, CME was significantly higher in the
antioxidant activity then those of SLE and ESE (p < 0.05). The antioxidant activity TPC of CME was significantly higher than those of SLE
and ESE (p < 0.05). Of the eight kinds of coffee beans, there was no distinctive trend in the highest and the lowest amounts of
antioxidant activity and TPC within each extraction method. CME was the most efficient method in extracting functional materials in
coffee which might be due to the higher amount of water used for extraction.

INTRODUCTION
Coffee, one of the major commodities being traded all over the
world (1), is considered as a hedonic food. Consumers tended
to focus on the favors of coffee rather than their functionality.
However, coffee contains significant amounts of functional
compounds such as caffeic, chlorogenic, hydroxycinnamic,
ferulic, sinapic acids (2-5), and melanoidins (6, 7) that have
strong antioxidant activity. Coffee is known as a major source
of chlorogenic acids for human diet (8, 9). Coffee is also known
as a major source of antioxidants in European countries (10, 11).
Many studies reported the functionality of coffee is influenced
by the degree of roasting (12), varieties (13), or roasting
methods (14). Although solid-liquid extraction (SLE) had been
used in many scientific studies (13-15), espresso extraction (ESE)
and coffee maker extraction (CME) are the most common
methods for extracting coffee. It was unknown whether the
antioxidant activity and total polyphenol content (TPC) of
coffee extracted by the SLE showed similar to the common
extraction methods in our real life to authors knowledge.
Therefore, the objective of this study was to compare the
antioxidant activity by two frequently used in vitro assays (DPPH
and FRAP) and TPC of eight different kinds of coffee beans
(Coffea Arabica L.) extracted by SLE, ESE and CME.

FeSO47H2O (Iron(II) sulfate heptahydrate), were purchased

from Sigma Aldrich -LLC. (St. Louis, MO, USA). 2, 4, 6-Tri
(2-Pyridyl)-1, 3, 5-triazine (TPTZ) was purchased from Tokyo
Chemical Industry Co. (Tokyo, Japan). Hydrogen chloride,
acetic acid and sodium acetate were purchased from
Samchun Chemical Co. (Seoul, Korea). Ethanol and methanol
were purchased from Junsei Chemical Co. (Tokyo, Japan).
Sodium carbonate was purchased from Showa Chemical
Industry Co. (Tokyo, Japan).

MATERIALS AND METHODS

Roasted coffee bean preparation

Eight coffee beans (Mexico Yajalon: MY, Mexico
Maragogype: MM, Guatemala Antigua: GA, Ethiopia Michile:
EM, Kenya AA: KA, Brazil Cerrado: BC, Colombia Supremo:
CS, Peru Chanchamayo: PC) harvested in 2013 were
purchased from local coffee bean traders (Moi, Yongin-si,
Korea; Beansguru Co., LTD., Seoul, Korea). Green coffee
beans (2000.0 g) were roasted using a commercial coffee
roasting machine (R-105, Fuji Royal Co., Ltd., Osaka, Japan)
by a coffee roasting specialist. Since the same roasting
condition was not necessary to produce consistent roasting
quality (13), different roasting conditions were applied based
on the condition of each coffee bean by the specialist to
maximize the quality of roasted coffee beans. The degree of
roasting was controlled by Agtron number (58 2) to meet
the light-medium roast of coffee (16). Roasted coffee beans
were kept at the room temperature for 72 hours for degassing.

Chemicals
Folin-ciocalteu reagent, gallic acid, 1,1-diphenyl-2picrylhydrazyl (DPPH), ascorbic acid, FeCl36 H2O and

Preparation of coffee extracts

The SLE was applied for extraction using slight modification
of Nebesny and Budryns method (14). The ratio between

Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016

15

the ground coffee and distilled water (98C) was 1:10 (w/v).
Extraction was carried out in a water bath at 98C for 10
min with 100 rpm in triplicates. Extracts were cooled with tap
water to 20C and centrifuged at 6,485 x g force for 15 min.
The supernatant was used for vacuum filtration. The ESE was
carried out using a commercial espresso machine (Faema
E98, Faema, Milan, Italy). Ground coffee beans (14.5 g) were
transferred to a porter filter and tampered. Approximately
60 mL of coffee was extracted. The CME was performed
using a home coffee maker (HD7564, Philips, Amsterdam,
Netherland). Coffee (10.0 g) was put into No. 2 coffee filters
(Melita USA Inc., Clearwater, FL, USA). Filtered water (200 mL)
was used for extraction. The extraction was continued for
4 min. All coffee extracts were vacuum-filtered through
Advantec No. 2 filter paper (pore size: 6 m, Advantec MFS,
Inc., Dublin, CA, USA). The extracts were frozen and stored in
a freezer (-30C) for analyses.
DPPH free radical scavenging activity
DPPH free radical scavenging activity was conducted using
modified Nebesny and Budryns method (14). Briefly, 160
L DPPH solution (1.4 x 10-4 M) and diluted coffee extract
(coffee extract: distilled water = 1: 100, v/v) were mixed
and kept at the room temperature for 30 min. The mixture
was added into a 96-well microplate and the absorbance
was measured using a microplate reader (Spectra max M2,
Molecular Devices, LLC. Sunnyvale, CA, USA) at 517 nm. A
blank was prepared by substituting the DPPH solution with
methanol. The difference in absorbance (Abssample Absblank)
was compared to the standard curve (R2 = 0.992) of the
absorbance of L-ascorbic acid solutions (0 56.25 g/mL).
DPPH free radical scavenging activity was expressed as mg
ascorbic acid equivalent/ g of ground coffee (mg ACE/g).
Ferric reducing antioxidant power (FRAP) assay
FRAP assay was conducted using modified Benzie and
Strains method (17). Briefly, FRAP reagent was made by
mixing 300 mM sodium acetate buffer (pH 3.6), 10mM
TPTZ/40 mM HCl, and 20 mM FeCl3 solution at ratio of 10:
1: 1 (v/v/v) and incubated at 37. The coffee extract
(10 L) and FRAP reagent (290 L) were mixed and kept
at 37 for 15 min. Their absorbance was measured at
593 nm using a microplate reader (Spectra max M2,
Molecular Devices, LLC). Standard curve (R2 = 0.997) by a
positive control (FeSO47H2O) was used to calculate the
FRAP value. Results were presented as mM
FeSO4/g of ground coffee.
Total polyphenol content
TPC were determined using Singletons
method (18). A 20 L of the extract was mixed
with distilled water (1,580 L). The mixture
(160 L) and 2-N Folin-Ciacalteus phenol
reagent (10 L) were mixed and kept for 8
min. After that, 30 L of Na2CO3 was added.
The mixture was kept at room temperature for
two hours. A blank sample was prepared by
substituting the reagent with distilled water.
After incubation, the absorbance values of
samples were measured with a microplate
reader (Spectra max M2, Molecular Devices,
LLC) at 765 nm against the blank sample.
The TPC was presented as mg gallic acid
equivalent/ g of coffee (mg GAE/g).

16

Statistical analyses
One-way analysis of variance (ANOVA) was conducted among
the eight coffee samples and three extraction methods. Fishers
least significant difference (LSD) test was conducted in order to
find out the differences for independent variables (samples or
extraction methods). Statistical significance was considered when
p-value was less than 0.05.

RESULTS AND DISCUSSION

The antioxidant activities of different coffee extracts determined
by DPPH assay are listed in Table 1. There were significant
differences (F2,71 = 68.20, p < 0.001) among the three extraction
methods. CME showed the highest antioxidant activity (average
of 26.70 ACE/g), followed by SLE (average of 23.91 ACE/g) and
ESE (average of 12.56 ACE/g). ESE contained approximately
half of the antioxidant activity compared to CME or SLE. For
different kinds of coffee beans, MY, MM, GA, BC, and CS were
not significantly different (p > 0.05) between CME and SLE. For
ESE extracts, each coffee variety had significantly lower (p < 0.05)
antioxidant activity compared to those of SLE or CME.
There were significant differences (p < 0.05) among samples
within each extraction method. The highest and lowest coffee
varieties for each extraction method were different. The highest
DPPH antioxidant activity of SLE, ESE, and CME were CS (25.83
mg ACE/g), MM (15.92 mg ACE/g), and EM (31.93 mg ACE/g),
respectively. The lowest DPPH antioxidant activity for SLE, ESE, and
CME were PC (20.90 mg ACE/g), GA (8.65 mg ACE/g), and MM
(23.09 mg ACE/g), respectively. The orders of antioxidant activities
within each extraction method were inconsistent.
The results of FRAP assay are shown in Table 2. FRAP assay results
showed different antioxidant activity pattern compared to DPPH
assay (Table 1). CME (average of 0.529 mM FeSO4/g) showed
the highest antioxidant activity, whereas SLE and ESE (average of
0.376 and 0.346 mM
FeSO4 / g, respectively) were not significantly different (F2,71 =
68.20, p > 0.05) each other. Inconsistent pattern of antioxidant
activity between FRAP and DPPH assays were also found
in Vignolis study (7). Therefore, different antioxidant activity
patterns from the three extraction methods could be due to the
different mechanism of antioxidant assays (19). The antioxidant
activity of coffee by FRAP assay was similar to black and Oolong
teas (20).

Table 1. DPPH free radical scavenging activity of eight roasted coffee bean extracted by
solid-liquid, espresso and coffee maker extractions.
1 Different superscripts within a column meant significant difference at p < 0.05 by Fishers
least significant difference (LSD) test. Different subscripts within a row meant significant
difference at p < 0.05 by Fishers LSD test.

Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016

When the antioxidant activity of different coffee extracted with

a certain extraction method was compared, different orders in
antioxidant activity of coffee varieties were found. The highest
antioxidant groups for each extraction method by Fishers LSD test
were EM (0.42 mM FeSO4/g) and KA (0.40 mM FeSO4/g) for SLE, KA
(0.41 mM FeSO4/g) for ESE, and PC (0.69 mM FeSO4/g) for CME.
The lowest antioxidant groups for each extraction method were
inconsistent. Interestingly, PC had the lowest antioxidant activities by
SLE and ESE, but PC had the highest antioxidant activity by CME.
TPC of coffee extracts using the three extraction methods are listed
in Table 3. The average amounts of TPC by SLE, ESE and CME were
26.11, 20.23, and 35.48 mg GAE/g, respectively. There were significant
differences (F2,71 = 71.60, p < 0.001) among the three extraction
methods. CME showed the highest amount of TPC in general. Only
EM showed the highest amount of TPC by SLE. The amounts of TPC
for each sample were significantly different. However, TPC among
samples in each extraction method did not show any pattern in
amount. The highest TPC were EM for SLE (34.23 mg GAE/g) and ESE
(21.99 mg GAE/g), and CS (47.29 mg GAE/g) for CME. PC was in the
lowest amount of TPC group for SLE and in the second lowest TPC
group for ESE. However, PC was in the second highest TPC group
in CME based on Fishers LSD test. PC showed lower antioxidant
activity and TPC in SLE and ESE, where as it belonged to the higher
antioxidant and TPC groups for CME. The TPC of coffee extracted
by SLE and ESE was lower than that from the black tea (30.5 mg
GAE/g) (21); however, CME showed higher TPC in 5 out of 8 coffee
beans. TPC was not strongly correlated with DPPH and FRAP assays
(r = 0.009 0.684) other than the correlation between TPC and FRAP
(r = 0.837) in ESE. Different origin of coffee beans would influence the
low correlation. The low correlation coefficients between antioxidant
activity and TPC were found in the previous coffee study (13).
Throughout the antioxidant activity and TPC, ESE showed the lowest
functionality. This trend was similar to a previous report in which
CME was significantly higher in deoxyribose damage than ESE (22).
Since ESE was extracted with lower amount of water, the functional
materials in coffee might have been insufficiently extracted. In
addition, ESE was carried out in a shorter time (30 sec) compared
to SLE (10 min) or CME (4 min). Therefore, ESE would not functional
materials from the ground coffee due to the much shorter time for
extraction. On the other hand, CME showed dominant functionality
in antioxidant activity and TPC. CME extraction was conducted with
1:20 (coffee: water, w/v) ratio, while SLE and ESE were conducted
with 1:10 and 1:4.14 ratios, respectively. The amount of water for each
gram of coffee might have significantly influenced the extraction of
functional materials.

CONCLUSIONS
Antioxidant activities and TPC of SLE were different from the two
regular coffee extraction methods, CME and ESE. In addition, the
orders of antioxidant activity and TPC of eight coffee varieties using
three extraction methods were different. Therefore, the antioxidant
activity and TPC by SLE from many previous studies might not reflect
the antioxidant activity and TPC of coffee extracts that consumers
have drunk from CME and ESE. Consumers consumed more
functional materials by CME but less functional materials by ESE in
comparison with SLE based on g of coffee. There was no obvious
trend in antioxidant activities and TPC among the eight coffee
varieties within each extraction method. This might be due to the
different inner structure and composition for each variety. Based on
the results of this study, CME appeared to extract more functional
materials from coffee beans.

Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016

7.

8.

9.

Table 2. Ferric reducing anxioxidant power (FRAP) of eight roasted coffee bean
extracted by solid-liquid, espresso and coffee maker extractions.
1 Different superscripts within a column meant significant difference at p < 0.05 by Fishers
least significant difference (LSD) test. Different subscripts within a row meant significant
difference at p < 0.05 by Fishers LSD test.

10.

11.

12.

13.

Table 3. Total polyphenol content of eight roasted coffee bean extracted by solid-liquid,
espresso and coffee maker extractions
1 Different superscripts within a column meant significant difference at p < 0.05 by Fishers
least significant difference (LSD) test. Different subscripts within a row meant significant
difference at p < 0.05 by Fishers LSD test.

ACKNOWLEDGEMENT
The authors thank to Jae Wook Do, owner of caf MOI, for
coffee roasting and espresso extraction.

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