LWT - Food Science and Technology 73 (2016) 243e250

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LWT - Food Science and Technology

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Relationship between antioxidant capacity, chlorogenic acids and

elemental composition of green coffee
Magdalena Jeszka-Skowron a, *, Ewa Stanisz a, Maria Paz De Pen
~a b
a  , Poland
Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, Berdychowo 4, 60-965 Poznan
b
Navarra Institute for Health Research, University of Navarra, E-31008 Pamplona, Spain

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

Article history: Antioxidant capacity of green coffee infusions (Arabica and Robusta of different geographical origin) has
Received 18 January 2016 been studied e.g. with the use of Folin-Ciocalteu assay (the average: 1043 ± 577 mg GAE/L). The highest
Received in revised form antioxidant capacity as well as concentration of three main 5-,4- and 3- caffeoylquinic acids were
5 June 2016
determined for Robusta coffee brews. According to assay used, steaming and decaffeination of Vietnam
Accepted 6 June 2016
Available online 7 June 2016
coffee beans altered the antioxidant capacity as well as the concentration of chlorogenic acids in the
infusions. Additionally, the method of Cd, Cu, Mn, Pb and Se determination in green coffee beans and
their infusions was optimized with the use of graphite furnace atomic absorption spectrometry. The
Keywords:
Green coffee infusion
average concentration of metals in green coffee beans was (mg/kg): Cd (0.013 ± 0.008), Cu (13.3 ± 2.8),
Elemental analysis Mn (24.1 ± 8.7), Pb (0.023 ± 0.005), Se (0.028 ± 0.010), respectively. The average leachabilities of Mn and
Chlorogenic acid Cu were established as 31.5% and 62.3%, respectively. The Cd, Pb and Se concentrations in the infusions
High performance liquid chromatography were below the limits of detections.
Antioxidant capacity assay © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Coffea canephora var. robusta belong to Rubiaceae family. Arabica

Food of plant origin contains many antioxidants and bioactive
compounds such as phenolics, vitamins/provitamins as well as
major, minor and trace elements. Higher intake of antioxidant-rich
products is associated with lower oxidative stress in the human
body. There are many evidences that diet rich in food antioxidants
especially from plants, herbs, spices and beverages shows protec-
tive effect on human health and reduces the risk of various diseases
(Benzie & Choi, 2014).
Popular beverages contain multitude of antioxidants and be-
tween them coffee brew showed high antioxidant capacity in
in vitro and in vivo tests (Daglia, Papetti, Gregotti, Berte, & Gazzani,
2000; Shahidi & Chandrasekara, 2010). Antioxidant capacity of
espresso coffee measured with the use of FRAP assay is even two
fold higher in comparison to other beverages such as red wine or
pomegranate juice (Carlsen et al., 2010). Coffee, prepared on filter
or boiled has the same capacity as red wine (Carlsen et al., 2010).
Coffee with unique aroma and flavour is one of the most popular
beverages in the world. Species of Coffea genus Coffea arabica and
* Corresponding author.
E-mail address: magdalena.jeszka-skowron@put.poznan.pl
Skowron).
usually comes from South America (i.e. Brazil) and from the up-
lands and mountain areas of East Africa. The main places of Robusta
origin are Vietnam and the lowlands of Central and West Africa as
well as South Asia (ICO, 2013).
Coffee contains antioxidants, mainly the chlorogenic acids
(caffeoylquinic acids). Green coffee extracts show a hypotensive
effect on rats (Suzuki, Kagawa, Ochiai, Tokimitsu, & Saito, 2002) and
reduce visceral fat and body weight (Igho, Rohini, & Edzard, 2011;
Shimoda, Seki, & Aitani, 2006). The other bioactive compounds
with antioxidant capacity such as caffeine, theophylline and theo-
bromine, tocopherols, cafestol, kahweol and trigonelline were also
determined (Jeszka-Skowron, Sentkowska, Pyrzyn  ska, & De Pen~ a,
2016; Jeszka-Skowron, Zgoła-Grzeskowiak, & Grzeskowiak, 2015;
Kuhnert et al., 2011; Perrone, Farah Donangelo, de Paulis, &
Martin, 2008).
Latest studies on chlorogenic acid (5-caffeoylquinic acid), the
main component of green coffee beans, showed the protective role
of this compound in neurons therefore it could prevent from
neurodegenerative diseases such as ischemic stroke (Mikami &
Yamazawa, 2015).
Antioxidant capacity of green coffee extracts of Arabica depends
on the calcium levels (Stelmach, Pohl, & Szymczycha-Madeja,
(M. Jeszka- 2015). Other trace elements such as copper, manganese and

http://dx.doi.org/10.1016/j.lwt.2016.06.018
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
244 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250
selenium also play a significant role in oxidative metabolism - they
are involved in redox processes that are essential for many meta-
bolic pathways as well as for cellular defence against oxidative
stress (Brigelius-Flohe, 2006). Simultaneously, low selenium di-
etary intake may result in higher cancer incidence and is associated
with development of cardiovascular diseases, cirrhosis, diabetes
and asthma (Fairweather-Tait et al., 2011; Navarro-Alarcon and
Lopez-Martinez, 2000).
Copper is a transition metal which distorts homeostasis and it
may induce oxidative stress and oxidative damage to proteins,
lipids and DNA (Festa & Thiele, 2011). Cadmium and lead, so-called
heavy or toxic metals, are most frequent contaminants in food
products and they have influence on the quality of coffee infusions.
The aim of the study was to compare the content of minor and
trace elements, three main chlorogenic acids (5-, 4- and 3-
caffeoylquinic acids) and antioxidant capacity of green coffee
Arabica and Robusta beans as well as infusions depending on the
place of origin and method of beans preparation (decaffeinated and
steamed Vietnam coffee beans). Folin-Ciocalteu (F-C), ABTS (2,2’-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays were used to
evaluate the antioxidant properties of coffee brews. High pressure
liquid chromatography (HPLC-UV-Vis) was used to determine 5-, 4-
and 3-caffeoylquinic acids in green coffee brews. Possible correla-
tions between the content of particular compounds and antioxi-
dant capacity were also in the scope of this study.
2. Materials and methods
2.1. Chemicals
2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) dia-
mmonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
chlorogenic acids (5-, 4- and 3-caffeoylquinic acids), gallic acid and
Folin-Ciocalteu reagent were purchased from Sigma-Aldrich
Chemical Co. (Steinheim, Germany). MS-grade acetonitrile, potas-
sium persulfate (di-potassium peroxdisulfate), and methanol were
obtained from POCH (Gliwice, Poland).
Working standard solutions of the elements were obtained by
appropriate dilution of the stock standard solutions (1000 mg/L
solution of Cd, Cu, Mn, Pb and Se in 0.5 mol/L nitric acid, Merck,
Darmstadt, Germany). All working standard solutions were pre-
pared daily; the appropriate stock solution was diluted with high-
purity water. Chemical modifier solutions: palladium 10.0 ± 0.2 g/
L Pd(NO3)2, magnesium 10.0 ± 0.2 g/L Mg(NO3)2 and phosphate
100.0 ± 2 g/L NH4H2PO4 were purchased from Merck. Ca. 14 mol/L
HNO3 and ca. 10 mol/L H2O2 of the highest quality (Suprapur,
Merck) were used for digestion of the samples. High purity water:
deionized and doubly distilled water (quartz apparatus, Bi18, Her-
aeus, Hanau, Germany) was also used throughout the experiments.
2.2. Material
The accuracy of the elements determination procedure was
studied using several certified reference materials (CRMs) with the
certified reference values of analytes (Cd, Cu, Mn, Pb and Se). The
following materials were chosen: SRM 1567a (Wheat flour), SRM
1549 (Nonfat milk powder) from the National Institute of Standards
and Technology (Gaithersburg, USA) and INCT-OBTL-5 (Oriental
Basma Tobacco Leaves), INCT-SBF-4 (Soya Bean Flour) and INCT-TL-
1 (Tea Leaves) from Institute of Nuclear Chemistry and Technology
(Warsaw, Poland). All solid reference materials were used as
bottled, without further grinding and sieving.
Fourteen green coffee samples from different regions of the
world were provided by Strauss Cafe  Poland (Swadzim/Poznan ,
Poland). It included Arabica green coffee beans from Brazil (TG),
Rwanda (Ordinary), China, Laos, Guatemala (SGH) and Peru (HB) as
well as Robusta green coffee beans from Uganda (Bugishu, SC12),
Indonesia, Vietnam (Gr2, decaffeinated Gr2, steamed Gr2), India
(Cherry) and Laos (FAQ). The geographical origin of the samples and
their types were confirmed by the supplier. The samples were
ground in a laboratory mill and digested without sieving (according
the procedure described in the section 2.4.1.)
2.3. Instrumentation for determination of elements and antioxidant
capacity
Determination of selected elements was performed with an AAS
5EA spectrometer (ET AAS) (Analytik Jena GmbH, Jena, Germany)
equipped with deuterium source background correction, a trans-
versely heated graphite atomizer and an MPE5 autosampler. Py-
rolytically coated graphite tubes were employed exclusively.
Appropriate hollow cathode lamps (Photron, Narre Warren,
Australia) were used as the radiation sources. Compressed argon of
UHP 5.5 purity obtained from Air Products (Warsaw, Poland) was
employed as a protective and purge gas. For wet digestion of
certified reference materials and coffee samples a UniClever
focused microwave sample preparation system (Plazmatronika,
Wrocław, Poland) operating at 2450 MHz and 300 W maximum
output was used. The computer-controlled system with continuous
temperature, pressure and microwave power monitoring was
equipped with high-pressure vessel made from modified PTFE
(poly(tetrafluoroethylene) modified with perfluoro(propylvinyl
ether) and water cooling system. The vessel capacity was 110 mL
and the maximum pressure and maximum temperature were
10.1 MPa and 300
C, respectively.
All spectrophotometric determinations connected with antiox-
idant capacity were performed with the use of Beckman UVeVis
Spectrophotometer 7500DU (Brea, CA, USA) with glass cuvettes of
1 cm length. Spectra were recorded in the range from 380 to
800 nm with 0.2 nm resolution. All determinations were carried out
in triplicate.
Coffee samples (beans) were ground in a laboratory mill IKA
(Staufen, Germany) A11 basic designed with a grinding chamber
mad of Tefcel (PTFE, glass fiber-reinforced) with stainless steel inlet.
2.4. Analytical procedures for elements determination
2.4.1. Microwave-assisted digestion of CRMs and coffee beans
samples
Approximately 0.500 g of powdered certified reference material
or ground coffee beans sample was placed in the vessel of the
microwave digestion system and moistened by 1 mL of 10 mol/L
H2O2. Then, 5 mL of 14 mol/L HNO3 were added. The sample was
heated for 20 min at 300 W. After digestion, the clear digested
solution was transferred into 10 mL calibrated flask and diluted to
volume with high-purity water. Before further analysis this solution
was appropriately diluted depending on the concentration levels of
the elements. A corresponding blank was also prepared according
to the above microwave-assisted digestion procedure. It was per-
formed to correct possible contamination from the reagents used
for the sample preparation.
2.4.2. AAS determination procedures
In the course of the study a graphite furnace was used for at-
omization of the analytes. In order to determine the elements in the
coffee beans samples after digestion, 20 mL (or 30 mL for Se deter-
mination) of the solution was injected into the graphite tube for ET
AAS determination under the optimized conditions. In order to
improve the removal of matrix without losing the analyte in the
 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250 245

pyrolysis stage the selected chemical modifiers were used (Table 1).
Calibration was performed by the method of standard additions.
The average blank value, if necessary, was subtracted from the
sample value after all calculations. Operating conditions of the ET
AA spectrometer used for determination of elements in coffee
beans after microwave-assisted digestion are shown in Table 1. The
concentration detection limits were defined as 3 times the standard
deviation of the blank signal (n ¼ 10) divided by the slope of each
calibration graph. LODs as well as the precision, expressed as RSD
(relative standard deviation (%); calculated for elements and each
sample) are shown in Table 1.
2.5. Analytical procedures for antioxidant capacity and chlorogenic
acids concentration
2.5.1. Extraction process
To prepare extracts (brews/infusions), analytes were extracted
from 0.500 g of ground beans by flooding with 20 mL of double
distilled water at temperature 95
C for 15 min. Then the solutions
were cooled to room temperature, centrifuged (5 min, 2038 g),
decanted, filtered through 0.45 mm polytetrafluoroethylene syringe
filter from Agilent Technologies (Santa Clara, CA, USA) and finally
diluted to proper volume with double distilled water (depends on
antioxidant capacity assay). All coffee extracts were prepared
directly before analysis. Folin-Ciocalteu, ABTS and DPPH assays
were used to evaluate the antioxidant capacity of the coffee
extracts.
2.5.2. Chlorogenic acids determination
The chlorogenic acids: 5-, 4- and 3-caffeoylquinic acids (5-CQA,
4-CQA, and 3-CQA) were determined using chromatographic sys-
tem equipped with analytical HPLC unit model 1100 (Agilent
Technologies, CA, USA) equipped with a binary pump and an
automated sample injector and UVeVis lamp (Agilent Technolo-
gies, CA, USA). 15 mL of samples were injected into a C18 column
(100 mm  2.1 mm I.D.; 2.6 mm; from Phenomenex, Torrance, CA,
USA) maintained at 25
C. The mobile phase employed in the course
of analysis consisted of 8 mmol/L formic acid in water (pH 2.8)
(solvent A) and acetonitrile (solvent B) at a flow rate of 0.8 mL/min.
Elution was initiated at 10% A and maintained for 5 min, the per-
centage of solvent A was increased to 20% in 10 min and maintained
for 10 min, then increased to 50% in 20 min and maintained for
3 min, and finally increased to 80% for 15 min. Detection was car-
ried out using a diode-array detector (DAD), and chromatograms
were recorded at 325 nm. Identification of 5-CQA, 4-CQA, and 3-
CQA was performed by comparing the retention times and the
photodiode array spectra with those obtained for reference
standard. Quantitation of chlorogenic acids were made by
comparing the peak areas with those of the standards.
2.5.3. DPPH antioxidant capacity
The ability of green coffee extracts to scavenge DPPH (2,2-
diphenyl-1-picrylhydrazyl) radicals was studied according to the
method reported previously by Jeszka-Skowron and Zgoła-
Grzeskowiak (2014). Briefly, coffee extract was diluted in meth-
anol (1:100; mL:mL), than 1.0 mL of 0.5 mmol/L methanol solution
of DPPH was mixed with 3 mL of the diluted extract. The mixture
was then mixed and left for 30 min at room temperature in the
dark. The absorbance of samples against a reagent blank was
measured at 516 nm. Antioxidant capacity was expressed as per-
centage of DPPH scavenging relative to control solution using the
following equation:
DPPH scavenging capacity ð%Þ ¼ ððAbsorbance of control
 Absorbance of sampleÞ
 =ðAbsorbance of controlÞÞ
 100%
2.5.4. Folin-Ciocalteu assay
Reducing ability of green coffee extracts was analyzed using
Folin-Ciocalteu’s reagent (Singleton, Orthofer, & Lamuela-Raventos,
1999). Gallic acid as an external standard was used (r2 ¼ 0.999).
Briefly 0.5 mL of sample (all coffee brews were diluted with water,
1:25; mL:mL) was mixed with 2.5 mL Folin-Ciocalteu’s reagent
(diluted with water, 1:10; mL:mL). After 4 min. 2.0 mL of 0.71 mol/L
Na2CO3 was added to this solution and mixed. The absorbance of
samples against a reagent blank was measured at 754 nm after 1 h
of incubation at room temperature in the dark. The results were
expressed as gallic acid equivalent (GAE) in mg/L of the brew.
2.5.5. ABTS antioxidant capacity
The ability of green coffee extracts to scavenge ABTS (2,2’-azi-
nobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)
radicals was studied according to the method of Re et al. (1999)
with a slight modification. Briefly, coffee extract was diluted in
water (1:5; mL:mL) than 3.0 mL of ABTSþ solution (prepared 24 h
earlier with 7 mmol/L ABTS and 2.45 mmol/L potassium persulfate)
was mixed with 0.03 mL of the diluted extract. The mixture was left
for 6 min at room temperature in the dark. The absorbance of
samples against a reagent blank was measured at 734 nm. Anti-
oxidant capacity was expressed as percentage of ABTS scavenging

Table 1
Operating conditions of the ET AA spectrometer used for determination of elements in CRMs and green coffee beans after microwave-assisted digestion.

Parameter Cd Cu Mn Pb Se

Wavelength (nm) 228.8 324.8 279.5 283.3 196.0

Spectral band width (nm) 0.8 0.8 0.2 0.5 1.2
Lamp current (mA) 4 3.0 3.5 8 10
Modifier NH4H2PO4 þ Mg(NO3)2 e Pd(NO3)2 NH4H2PO4 Pd(NO3)2
Modifier concentration (mg/L) 1c þ 0.6c e 2 20 2
Modifier volume (mL) 4 e 5 5 5
Sample volume (mL) 20 20 20 20 30
Pyrolysis temp. (
C)/hold (s) 850/4 700/2 1100/2 900/2 350 and 1050d/20 and 10
Atomization temp. (
C)/hold (s) 1650/2 1800/4 2200/2 1800/4 2100/4
LODa (ng/mL) 0.11 0.07 0.02 0.11 0.18
Precisionb (%) 8e13 3e7 4e7 8e13 9e13
a
Limit of detection calculated according to a 3s blank criterion (the standard deviation of the blank, n ¼ 10).
b
As relative standard deviation (RSD) for 5 replicated measurements of samples solutions containing Cd, Cu, Mn, Pb and Se.
c
g/L.
d
Two step pyrolysis was used.
246 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250

relative to control solution using the following equation:
ABTS scavenging capacity ð%Þ: ¼ ððAbsorbance of control
 Absorbance of sampleÞ
 =ðAbsorbance of controlÞ Þ
 100%
Trolox was used as a standard for the calibration curve. The ABTS
scavenging activity was presented as Trolox equivalents (TE) using
the following linear equation (r2 ¼ 0.999). The results were
expressed as mmol TE/L of the infusion.
2.6. Statistical analysis
Results are expressed as average ± standard deviation (at least
three replicates). Analysis of variance and significant differences
among averages and correlation analysis were performed with one
way ANOVA. The significance level was based on a confidence level
of 95.0%. The experimental data were analyzed using Statistica 10.0
program (StatSoft Inc., Tulsa, OK, USA). The reproducibility of the
results was expressed as pooled standard deviation values (Pooled
SD) calculated as a square root of a sum of individual variances
pondered by the individual degrees of freedom of each series of the
replicates.
3. Results and discussion
3.1. Chlorogenic acids concentration and antioxidant capacity of
green coffee extracts
Main chlorogenic acids (5-CQA, 4-CQA, and 3-CQA) were esti-
mated by the chromatography analysis. The average concentration
of these compounds in Arabica and Robusta coffee brews was:
176 mg/L and 153 mg/L, respectively (Table 2). The highest con-
centration of three main chlorogenic acids among Robusta coffee
type was obtained for coffee from Vietnam Gr2 decaffeinated e
221 mg/L of brew. It could be due to the solvent used and moisture
of beans after the processes (steaming or decaffeination of coffee
beans). Steaming of Robusta coffee is a method improving and
creating the specific acidic taste and flavour unique for Arabica
coffees. It was found that steaming of Vietnam GR2 coffee
decreased 5-CQA content (51%) but increased 4- and 3-CQA in
comparison to Vietnam GR2 coffee brew (43% and 63%,
respectively).
The significant differences between Arabica and Robusta coffee
brews concentration of 4- and 3-CQA was found: 31 ± 4 mg/L and
26 ± 4 mg/L for 4-CQA; 16.2 ± 0.8 and 20.3 ± 0.9 for 3-CQA (Tukey’s
test; p  0.05).
The concentration of three main chlorogenic acids: 5-CQA, 4-
CQA, and 3-CQA in the studied Arabica coffee brews were in the
range of 132e164 mg/L (Table 2). These results were similar to
these published previously (Ky et al., 2001).
Antioxidant capacity of the coffee brews was measured by ABTS,
DPPH and Folin-Ciocalteu assays based on a single-electron transfer
reaction. These assays are widely used to determine in vitro anti-
oxidant capacity of foods and beverages (Pellegrini et al., 2006;
Stelmach et al., 2015).
Coffee infusions prepared from green beans of Robusta showed
higher antioxidant capacities than Arabica extracts (Table 3).
Arabica coffee from Peru showed the highest antioxidant capacity
in Folin-Ciocalteu and DPPH assays in comparison to others Arabica
coffees.
Coffee extract from Laos (Robusta) possessed 2-fold lower
antioxidant capacity in DPPH assay than other Robusta extracts and
even lower than Laos (Arabica). Laos coffee extract from Arabica
showed the lowest antioxidant capacity in almost all tests (Table 3).
Steaming and decaffeination of Vietnam GR2 coffee increased
antioxidant capacity in ABTS but decreased capacity in Folin-
Ciocalteu and DPPH assays in comparison to Vietnam GR2 coffee
infusion.
Antioxidant capacities of Arabica and Robusta green coffees
were on average 1043 mg GAE/L, 47.9 mmol TE/L and 56.3% in
antioxidant capacity assays: Folin-Ciocalteu, ABTS and DPPH assays,
respectively (Table 3). These results were similar to findings re-
ported by Naidu, Sulochanamma, Sampathu, and Srinivas (2008)
and Stelmach et al. (2015). Instead, the results of ABTS assay were
higher for these performed for green coffee brews than the bev-
erages prepared from the roasted beans such as espresso coffees
(26.96e36.54 mmol TE/L) (Pellegrini et al., 2003).
The biggest differences between Arabica and Robusta coffees
were found for Folin-Ciocalteu assay, probably due to higher level

Table 2
Chlorogenic acids (5-, 4- and 3-caffeoylquinic acids) content in Arabica and Robusta coffee brews (mg/L).

Coffee 5-Caffeoylquinic acid 4-Caffeoylquinic acids 3-Caffeoylquinic acid

Arabica Brazil TG 117c 19.7a 14.9a

Rwanda Ordinary 122d 21.4b 16.7b
China 115c 20.5ab 16.3b
Laos 97a 19.1a 15.8ab
Guatemala SGH 124de 20.2a 16.3b
Peru HB 126e 21.1b 17.2b
Pooled SD 1.3 0.7 0.6
Robusta Vietnam Gr2 110b 27.3d 21.8d
Vietnam Gr2 steamed 56 39.2 35.4
Vietnam Gr2 decaf. 104 59.8 57.2
India Cherry 137e 24.0c 18.9c
Laos FAQ 144g 22.1b 16.5b
Indonesia 126e 32.1e 27.2e
Uganda SC12 136f 24.7c 19.3c
Uganda Bugishu 135f 20.9ab 15.0a
Pooled SD 1.4 0.8 0.8
Min. 56 19.1 14.9
Max. 144 59.8 57.2
Average 118 26.6 22.0

Average value (n ¼ 3).

Averages in a column with different letters are significantly different (Tukey’s test; p  0.05); Processed coffees (Vietnam Gr2 steamed and Vietnam GR decaf) were not taken
into account in the statistical analysis as process is an additional parameter.
 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250 247

Table 3
Antioxidant capacity of coffee brews.

Coffee F-C (mg GAE/L) ABTS (mmol TE/L) DPPH (%)

Arabica Brazil TG 385ab 37.9b 47.5b

Rwanda Ordinary 434ab 36.2b 45.7b
China 394ab 38.7b 49.8bc
Laos 354a 33.7b 31.9a
Guatemala SGH 418b 27.3a 53.5c
Peru HB 494c 36.0b 59.8d
Pooled SD 27 0.6 2.1
Robusta Vietnam Gr2 1700i 57.1d 72.6g
Vietnam Gr2 steamed 1665 67.6 68.2
Vietnam Gr2 decaf. 1623 63.4 63.9
India Cherry 1552g 59.9de 63.2de
Laos FAQ 1458f 55.0cd 36.3b
Indonesia 1442f 48.7c 68.6fg
Uganda SC12 1441e 61.0e 65.6ef
Uganda Bugishu 1271d 48.3c 62.3de
Pooled SD 28 1.6 1.0
Min. 354 27.3 31.9
Max. 1700 67.6 72.6
Average 1043 47.9 56.3

Average value (n ¼ 3).

Averages in a column with different letters are significantly different (Tukey’s test; p  0.05); Processed coffees (Vietnam Gr2 steamed and Vietnam GR decaf) were not taken
into account in the statistical analysis as process is an additional parameter.

of chlorogenic acids in Robusta coffees - total chlorogenic acids
content: 176 mg/L (average for Robusta coffees) to 153 mg/L
(average for Arabica coffees). These findings were also confirmed by
Perrone, Farah, Donangelo, de Paulis, and Martin (2008).
The positive correlation between Folin-Ciocalteu and ABTS as-
says (r2 ¼ 0.862) with the use of Pearson’s linear correlation coef-
ficient was found. There was no significant correlation between
Folin-Ciocalteu and DPPH assays (r2 ¼ 0.458) and ABTS and DPPH
(r2 ¼ 0.3541) assays. Other authors reported the same similarity in
correlation between Folin-Ciocalteu and ABTS assays (Perez-
Hernandez, Chavez-Quiroz, Medina-Juarez, & Meza, 2012); but
different in Folin-Ciocalteu and DPPH assays (Stelmach et al., 2015).
No correlation between concentration of 5-, 4- and 3-CQA and
antioxidant capacity was found - especially in steamed coffee brew
(Vietnam origin). After the steaming process chlorogenic acids
content is lower. However, other compounds such as Maillard re-
action products probably are formed, as in the roasted coffee beans.
These compounds also possess the antioxidant capacities (Perez-
Hernandez et al., 2012).
The most commonly used methods for the measurement of
antioxidant capacity are ABTS and DPPH assays based on scav-
enging of stable free radicals by antioxidants. These methods differ
in terms of kinetics and experimental conditions. The ABTSþ
radical scavenging assay is related to both lipophilic and hydro-
philic antioxidants (Re et al., 1999). Ozgen, Reese, Tulio, Scheerens,
and Miller (2006) suggest that the DPPH assay is more suitable for
samples with lipophilic antioxidants or those characterized by a
high lipid content. Phenols are hydrophilic, and this may be the
reason why the differences in antioxidant capacity of coffee sam-
ples were observed. Therefore, it may be related to the solubility of
phenols.
The Folin-Ciocalteu method is used to evaluate reducing ability
of antioxidants. It has been found the most appropriate for this
purpose, as compared to the other methods (Singleton et al., 1999).
The Folin-Ciocalteu assay relies on the transfer of electrons in
alkaline medium from phenolic compounds to phosphomolybdic/
phosphotungstic acid complexes. The blue complexes formed are
determined with the use of spectrophotometric technique at
approximately 760 nm.
However, these methods cannot be translated to in vivo activ-
ities but can compare activities between samples.
3.2. Concentration of Cd, Cu, Mn, Pb and Se in green coffee beans
and their infusions
The elements that are usually determined in coffee beans can be
divided in the tree main groups (Pohl, Stelmach, Wełna, &
Szymczycha-Madeja, 2013). First one, named major elements or
macronutrients, consist of Ca, K, Mg, Na, S and P. Minor elements (or
micronutrients): Cl, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Sr and Zn are
classified in the second group. The analytes such as: Al, As, B, Ba, Br,
Cd, Hg, Pb and Sn consisted the group of trace elements (Pohl et al.,
2013).
Due to the fact that there were no available CRMs compatible
with the samples matrix, to study the reliability of the results, CRMs
of other plant or food samples have been used. This kind of mate-
rials is supposed to represent the coffee matrix the best (Pohl et al.,
2013). The precision and accuracy of the ET AAS method was
studied by analyzing five certified reference materials with
different matrices (Table 4): wheat flour (SRM 1567a) (Tagliaferro,
De Nadai Fernandes, & Bacchi, 2006), milk powder (SRM 1549)
(Suseela, Bhalke, Vinod Kumar, Tripathi, & Sastry, 2001; Ribeiro,
Moretto, Arruda, & Cadore, 2003), tea leaves (INCT-TL-1)
(Tagliaferro et al., 2006); Tagliaferro, De Nadai Fernandes, Bacchi,
Bode, & Joacir De França, 2007; Fran  kova
, Drabek, Havlík,
Szakova , & Vane
k, 2009) as well as tobacco leaves (INCT-OBTL-5)
and soya flour (INCT-SBF-4). The results obtained for digested CRMs
were in a good agreement with certified values for CRMs according
to the t-test at a 95% confidence level (Table 4). Recoveries of the
elements determined were: 95e117% for Cd, 102e106% for Cu,
98e104 for Mn, 110e111% for Pb and 91% for Se. Precision for
reference materials (express as the relative standard deviations,
RSD) was in the range from 4% to 19%.
The concentrations of selected elements in coffee beans were
determined and the analytical results, as averages of five de-
terminations with standard deviations around average values, are
given in Table 5. The determination of elements was conducted in
the samples after microwave-assisted digestion.
The lowest concentration was established for trace element -
cadmium, with average value of 0.013 ± 0.008 mg/kg in all samples.
Its concentrations varied form 0.008 mg/kg in Arabica from
Guatemala and Ugandan Robusta to 0.036 mg/kg in Chinese
Arabica. Cadmium contents in Arabica coffees were slightly higher
248 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250

Table 4
Determination of selected elements in CRMs with the use of ET AAS technique.

Element (mg/kg) SRM 1567a SRM 1549 INCT-OBTL-5 INCT-SBF-4 INCT-TL-1

Wheat flour Nonfat milk powder Oriental Basma tobacco Soya bean flour Tea leaves
leaves

Found Certified Found Certified Found Certified Found Certified Found Certified

Cd e ea e ea 2.51 ± 0.19 2.64 ± 0.14 e eb 0.035 ± 0.005 0.030 ± 0.004

Cu e ea e ea e ea 15.11 ± 0.85 14.30 ± 0.46 20.9 ± 1.2 20.4 ± 1.5
Mn e ea e ea 176 ± 7 180 ± 6 33.5 ± 1.6 32.3 ± 1.1 e ea
Pb e eb 0.021 ± 0.004 0.019 ± 0 .003 e ea e eb 1.95 ± 0.19 1.78 ± 0.24
Se 1.0 ± 0.2 1.1 ± 0.2 0.10 ± 0.02 0.11 ± 0.01 e eb e eb e eb
a
Not determined (the determination has not been conducted).
b
Value not available in the CRM.

Table 5
Concentration of Cd, Cu, Mn, Pb and Se in green coffee beans and their infusions.

Coffee Element1, beans (mg/kg) Element1, infusions5 (mg/L)

Cd Cu Mn Pb Se Cu Mn

Arabica Brazil TG 0.011b 12.5c 35.2d 0.019b 0.032b 0.168c 0.224h

Rwanda Ordinary <LOD2 13.6cd 19.2c 0.027c 0.028b 0.175d 0.280j
China 0.036d 8.4a 116f 0.019b 0.021a 0.205f 0.750l
Laos <LOD 11.9bc 37.1de 0.013a 0.031b 0.161b 0.332k
Guatemala SGH 0.008a 10.7b 20.3c 0.027c 0.029b 0.182de 0.180f
Peru HB 0.011b 11.7bc 40.3e 0.025c 0.019a 0.146a 0.190g
Pooled SD 0.002 0.7 2.4 0.001 0.003 0.004 0.009
Robusta Vietnam Gr2 0.012bc 11.9bc 20.9c 0.020b 0.031b 0.186e 0.160e
Vietnam Gr2 steamed 0.009 12.8 26.8 0.028 0.018 0.115 0.244
Vietnam Gr2 decaf. 0.012 17.5 27.1 0.023 0.012 0.181 0.176
India Cherry 0.015c 14.6d 16.7b 0.030d 0.046c 0.229g 0.100b
Laos Faq 0.011b 19.4f 16.3b 0.025c 0.021a 0.344j 0.124d
Indonesia 0.012bc 16.0de 17.6b 0.022b <LOD 0.203f 0.052a
Uganda SC12 0.008a 13.3cd 12.3a 0.025c 0.047c 0.292i 0.116c
Uganda Bugishu 0.009a 11.5bc 22.9c 0.019b 0.025ab 0.247h 0.254i
Pooled SD 0.002 0.9 1.3 0.001 0.003 0.007 0.005
Min. 0.008 8.4 12.3 0.013 0.012 0.115 0.052
Max. 0.036 19.4 116 0.030 0.047 0.344 0.750
Average 0.013 13.3 30.63/24.14 0.023 0.028 0.202 0.2273/0.1874
1
Average value (n ¼ 3)
2
Below limit of detection (LOD) (see Table 1).
3
Average value with coffee beans/infusion from China.
4
Average value without coffee beans/infusion from China.
5
Infusions were prepared by flooding the ground beans (0.500 g) with 20 mL of double distilled water at temp. 95
C for 15 min.
Averages in a column with different letters are significantly different (Tukey’s test; p  0.05); Processed coffees (Vietnam Gr2 steamed and Vietnam GR decaf) were not taken
into account in the statistical analysis as process is an additional parameter.

than in Robusta beans: 0.017 ± 0.013 mg/kg and 0.011 ± 0.002 mg/
kg, respectively.
Cadmium as well as lead and selenium are rarely determined in
coffee beans compared to other major and minor elements. This is
due to the fact that their concentrations in coffee are relatively low
and thus low limits of detection are required (Ashu &
Chandravanshi, 2011).
Very low variation in the contents was found for Se and Pb, with
average contents in all samples equal to 0.028 ± 0.010 mg/kg and
0.023 ± 0.005 mg/kg, respectively. There was also no significant
differences in the average content of these elements in Arabica and
Robusta coffees.
Cadmium and lead were determined below the maximum
allowable limit specified by the Polish Food Legislation. The values
defined by the Polish Food Legislation for Cd and Pb are 0.10 mg/kg
and 1.00 mg/kg, respectively.
The detected selenium levels were relatively low, with the
average value 0.028 mg/kg. Uganda Robusta SC12 and India Cherry
Robusta contain greater amounts of Se (0.047 mg/kg and 0.046 mg/
kg, respectively) as compared to the rest of the varieties of beans
ranging from 0.012 mg/kg to 0.032 mg/kg. The results obtained are
comparable to those published by Zaidi, Fatima, Arif, and Qureshi
(2006). Zaidi et al. (2006) also determined the higher concentra-
tion of selenium in the Indian coffee beans (0.100 mg/kg) compared
with coffee from other countries (Brazilian (0.014 mg/kg) and
Caribbean coffee (0.027 mg/kg)). Barbosa et al. (2014) found that
the concentration of Se values was in the range 0.0824e0.309 mg/
kg for conventional coffees and the concentration of Se in organic
coffees was in the range 0.0471e0.133 mg/kg.
The concentration of selenium in steamed and decaffeinated
coffee beans from Vietnam Gr2 (Robusta) were lower in compari-
son to untreated Vietnam Gr2 beans, 42% and 61% respectively.
For cadmium and lead, the concentrations found were lower or
stayed in accordance with the values reported in the literature.
Other Authors have determined cadmium concentrations in green
coffee as 0.70e0.75 mg/kg (dos Santos, dos Santos, Conti, dos
Santos & de Oliveira, 2009) and lead concentrations below
0.01 mg/kg (dos Santos et al., 2009). In roasted and ground coffees
Suseela et al. (2001), Grembecka, Malinowska, and Szefer (2007),
Santos et al. (2008), Ashu and Chandravanshi (2011) detected the
cadmium values as 0.001e0.1 mg/kg and lead values announced by
Suseela et al. (2001), Grembecka et al. (2007), Santos et al. (2008),
Ashu and Chandravanshi (2011) were in the range 0.021e2.6 mg/
kg. Barbosa et al. (2014) found that the concentration of lead values
 M. Jeszka-Skowron et al. / LWT - Food Science and Technology 73 (2016) 243e250
was in the range 0.029e0.29 mg/kg (for conventional coffees) and
in the range 0.016e0.165 mg/kg (for organic coffees); cadmium
concentration in conventional coffees was in the range
0.0001e0.011 mg/kg and the concentration of this element in
organic coffees was in the range 0.001e0.009 mg/kg.
The concentrations of copper did not present a major difference
between the samples. The average Cu value determined for all
samples was 13.3 ± 2.8 mg/kg with 11.5 ± 1.8 mg/kg and
14.6 ± 2.8 mg/kg for Arabica and Robusta coffees, respectively. For
Mn the average value determined for all samples was
30.6 ± 26.0 mg/kg. The largest difference was observed between
average Mn concentrations in Arabica (44.7 ± 36.2 mg/kg) and
Robusta coffees (20.1 ± 5.3 mg/kg). Arabica from China stood out for
the highest concentration of Mn - 116.3 ± 5.2 mg/kg (the average
for Arabica without this value was 30.4 ± 9.9). On the other hand
Robusta is characterized by relatively higher contents of Cu in
comparison with Arabica. Mn content seems to be appropriate to
characterize the Arabica and Robusta varieties. These observations
are in a good agreement with other reports (Martín, Pablos, &
Gonz alez, 1998).
The determination of Cd, Cu, Mn, Pb and Se was also conducted
in infusions obtained during extraction procedure. The Cd, Pb and
Se concentrations were below the limits of detections (see Table 1).
Relatively low variation in the concentrations was found for Mn and
Cu with average contents in all infusions equal to 0.227 ± 0.187 mg/
L and 0.202 ± 0.060 mg/L, respectively. Also in the case of infusions,
Chinese Arabica extract stood out for the highest concentration of
Mn (0.750 mg/L), the average for infusions without this value was
0.187 ± 0.079 mg/L. Manganese concentrations varied much more
than copper ones, form 0.052 mg/L in Indonesia Robusta extract to
0.750 mg/L in Chinese Arabica. Cu contents varied form 0.115 mg/L
in steamed Robusta Gr2 extract to 0.344 mg/L in Laos Faq Robusta.
The leachability of the metal which has been leached into the
coffee solution during brewing process, was expressed as a per-
centage (%). It was calculated on the basis of the determined
amount of element in the ground coffee beans and its concentra-
tion in infusion (the mass of coffee taken for the infusion and the
volume of the resulting extract were also considered). The calcu-
lated average leachabilities of Mn and Cu were established as 31.5%
and 62.3%, respectively. In the case of Mn the leachability was lower
compared to this reported by Stelmach et al. (2015) and more
similar to Ashu and Chandravanshi (2011) results obtained for
roasted Ethiopian coffee infusions. For Cu established value was
more similar to the findings of Stelmach et al. (2015) but much
more higher in comparison to roasted Ethiopian coffee infusions
studied by Ashu and Chandravanshi (2011). The leachability of Mn
was established to change from 11.8% (for Indonesian Robusta) to
58.3% (for Rwanda Ordinary Arabica). A higher leachability was
calculated for Cu, values varied from 35.9% for steamed Robusta
from Vietnam to 97.6% for Chinese Arabica.
Pearson’s correlation coefficient between antioxidant capacity
and elements in coffee brews was analyzed. There was found linear
correlation between concentration of Cu and Mn in Arabica coffee
brews (r2 ¼ 0.5683). This result is in agreement with other Authors
(Stelmach et al., 2015). There was also found linear correlation
between concentration of Cu and chlorogenic acids of Robusta
Vietnam coffee brews (r2 ¼ 0.4847). These correlations may be
important for human health because these compounds are
involved in antioxidative defense system of the organism. The
chlorogenic acids with other phenolic acids and flavonoids are the
enzymatic antioxidants. Copper and manganium are the part of
endogenous antioxidants and radical scavengers such as superox-
ide dismutase (SOD1, SOD2 and SOD3) (McCord & Fridovich, 1988).
249
4. Conclusions
Among coffees of different origin antioxidant capacity of Viet-
nam Robusta coffee brew was the highest. The processes of the
treating coffee beans during its production altered the antioxidant
capacity (coffee from Vietnam), therefore decaffeinated and
steamed coffees possessed higher antioxidant capacity in ABTS
assay. These coffee brews contain the higher concentration of Mn.
The processes also affected the concentration of 5-, 4- and 3-
caffeoylquinic acids in Vietnam coffee brews. To the best of our
knowledge, this work is the first report on the direct influence of
the decaffeination and steaming processes on the changes in the Se,
Cu, and Mn concentrations in green coffee beans.
Green coffee brews possess high antioxidant capacity and they
are rich in exogenous antioxidants such as 5-, 4- and 3-CQA. Other
elements such as Mn, Cu, or Se may play a role in endogenous
antioxidants such as superoxide dismutase or Se-glutathione
peroxidase.
Notes
The authors declare no competing financial interest.
Acknowledgement
This article was financially supported within the project 03/31/
DSPB/0316.
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