Food Chemistry 166 (2015) 261269

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Coffee with ginger Interactions of biologically active phytochemicals

in the model system
Agata Durak a,, Urszula Gawlik-Dziki a, Iwona Kowlska b
a
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland
b
Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich Str. 8, 24-100 Pulawy, Poland

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

Article history: This study evaluated the interactions between antiradical and anti-inammatory compounds from coffee
Received 6 March 2014 and ginger. Results obtained for whole plant material extracts were compared with those for chlorogenic
Received in revised form 6 May 2014 and caffeic acids (the main hydroxycinnamic acids of plant material). All the tested samples showed the
Accepted 17 June 2014
ability to scavenge free radicals and to inhibit lipoxygenase (LOX) activity. Both of these activities
Available online 24 June 2014
increased after simulated gastrointestinal digestion. Aromatic additives, such as ginger, are able to
change the antioxidant properties of coffee extract and antioxidant interactions may be identied using
Keywords:
two methods. Antiradical phytochemicals from coffee and ginger acted synergistically isoboles adopted
Coffee
Ginger
a concave form, while after digestion in vitro an additive reaction was observed; in turn, chemical stan-
Isobolographic analysis dards acted antagonistically. Water extractable LOX inhibitors acted antagonistically; however, after
Interaction factor digestion in vitro synergism was observed. The same kind of interaction was determined for standard
Antiradical activity compounds. These results were conrmed by IF (interaction factor) analysis.
Anti-inammatory activity 2014 Elsevier Ltd. All rights reserved.

1. Introduction inammation via deactivation of a range of pro-oxidative enzymes,

The oxidation of polyunsaturated fatty acids in biological mem-
branes leads to serious damage such as coronary atherosclerosis,
emphysemas, cancer and cirrhosis. Safeguarding fats against oxi-
dation is normally effected by restricting the access of oxygen or
adding antioxidants. The most commonly applied antioxidants
are synthetic phenols, such as butylated hydroxytoluene and
butylated hydroxyanisole (BHA). Their safety, however, has been
questioned (Imadia et al., 1983). Therefore, natural antioxidants
are readily acceptable by consumers, as they are considered safe.
Unlike their synthetic counterparts which are faced with tight gov-
ernmental legislation (Hancock & Viola, 2005), antioxidants from
natural sources are identical to the food that people consume reg-
ularly or use as condiments and they play important roles in the
human diet. Antioxidants act as free radical-scavengers and inhibit
lipid peroxidation and other free radical-mediated processes;
thereby helping to protect the human body from several diseases
attributed to the reactions of radicals (Atoui, Mansouri, Boskou, &
Kefalas, 2005). Reactive oxygen species generation during inam-
mation is bound, inter alia, with lipoxygenase (LOX) activity.
Phenolic plant compounds have been suggested as inhibitors of
Corresponding author. Tel.: +48 81 4623329; fax: +48 81 4623324.
E-mail address: agatadurak@gmail.com (A. Durak).
including inhibition of LOX-mediated arachidonic acid metabolism
(Gawlik-Dziki et al., 2012).
Plants such as herbs have long been used in traditional/folk
medicine in various cultures throughout the world. Zingiber ofci-
nale is one such traditional folk medicinal plant that has been used
for over 2000 years (Tepe, Sokmen, Akpulat, & Sokmen, 2006). The
rhizome of ginger is known for its contribution to food and has
antioxidant (Ghasemzadeh, Jaafar, & Rahmat, 2010) and antimicro-
bial potentials (Martins et al., 2001). In addition to its aromatic
contribution to foods, ginger has been reported to improve blood
circulation, reduce blood glucose levels in diabetic patients, aid
digestion, and is used in the treatment of nausea (Ernst & Pittler,
2000; Riddell & Perkins, 2009). Ginger is an indispensable compo-
nent of many food additives. With regards to antioxidant proper-
ties, it can be successfully used as a component in curry powder,
sauces, gingerbread and ginger-avoured carbonated drinks and
also in the preparation of dietaries for its aroma and avour. Many
authors discuss the chemical composition of ginger (Bartley &
Jacobs, 2000; Nishimura, 1995), but no toxic effect has been found.
In recent years, due to increased interest in nding physiologi-
cally functional foodstuffs, the relationship between coffee and
health has been extensively studied (Higdon & Frei, 2006). Antiox-
idant activity in foods and beverages is one of the properties that
has generated much interest within the scientic community

http://dx.doi.org/10.1016/j.foodchem.2014.06.075
0308-8146/ 2014 Elsevier Ltd. All rights reserved.
262 A. Durak et al. / Food Chemistry 166 (2015) 261269
(Yukawa et al., 2004). In coffee, antioxidant activity is generally
associated with the levels of indigenous phenolic compounds con-
tained therein, as well as Maillard reaction products, the latter
being generated during roasting (Del Castillo, Ames, & Gordon,
2002). Among the different phenolic compounds in coffee, the
most abundant are hydroxycinnamic acids which exist mainly in
esteried form. The best example is chlorogenic acid (5-caffeoyl-
quinic acid) (CGA) with an average level of 100 mg per cup of cof-
fee (Clifford, 1999). Few free phenolic acids are present in coffee,
although small quantities of caffeic, ferulic, and vanillic acids have
been detected (Drea & da Costa, 2005).
In contrast to synthetic pharmaceuticals based upon single
chemicals, many phytomedicines exert their benecial effects
through the additive or synergistic action of several chemical com-
pounds acting at single or multiple target sites associated with a
physiological process (Williamson, 2001). The method used for
the identication of interactions between active compounds is
isobolographic analysis. This method is independent of any activity
mechanism; however, it should be emphasised that this analysis is
quite complicated and labour-intensive. A denitely less compli-
cated method for the determination of interactions between
mixture components interaction factor (IF) was proposed by
Gawlik-Dziki (2012). This way of studying interactions, as with
isobolographic analysis, is independent of the mechanism of an
activity and requires a linear relationship between an activity
and sample concentration. Moreover, the strength of an interac-
tion may be approximately estimated based on the IF value.
There have been reports on the antioxidant activities of coffee
compounds (Gomez-Ruiz, Leake, & Ames, 2007) and ginger
(Ghasemzadeh et al., 2010). However, there is limited information
on the antioxidant property of coffee with ginger as an aromatic
supplement, and its potential for the management of oxidative
stress-related metabolic disorders. Therefore, this work reports
on a preliminary investigation into the antioxidant and anti-
inammatory properties of hot water extracts of coffee and ginger
mixtures and their potential as functional food.
Thus, the aim of this study was to test two hypotheses: (1) that
interactions between bioactive phytochemicals play a crucial role
in the creation of the nutraceutical potential of coffee fortied with
ginger, (2) that interaction with the food matrix and/or changes
during simulated gastrointestinal digestion cause signicant dif-
ferences in the relationship between the main hydroxycinnamic
acids contained in extracts compared to pure chemicals (standard
compounds).
2. Materials and methods
2.1. Chemicals
ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid),
a-amylase from hog pancreas (50 U/mg), pepsin from porcine gas-
tric mucosa (250 U/mg), pancreatin from porcine pancreas, bile
extract, lipoxygenase, linoleic acid, PBS (phosphate buffered saline)
were purchased from SigmaAldrich company. Acetonitrile and
methanol gradient HPLC grade and formic acid LCMS grade for
LCUVMS separations were purchased from J.T. Baker (Phillips-
burg, New Jersey, USA). Water was puried in-house with a
Milli-Q water purication system Simplicity-185 (EMD Millipore
Corporation, Billerica, Massachusetts, USA). All other chemicals
were of analytical grade.
2.2. Materials
The experimental material consisted of ground coffee available
on the Polish market, typical average quality coffee. Ginger was
bought from a local market (Lublin, Poland; Manufacturer: Tchibo
GmbH, Hamburg, Germany) in the form of ground spice.
The coffee brew and the ginger brew the aromatic supple-
ment, were analysed separately and in appropriate combinations
(4:1; 3:2; 1:1; 2:3; 1:4 v/v) coffee/ginger.
Model compound solutions were prepared in water and the
nal concentration was 10 lg/mL. The chlorogenic acid solution
and the solution of caffeic acid were analysed separately and in
appropriate combinations (4:1; 3:2; 1:1; 2:3; 1:4 v/v) chlorogenic
acid/ caffeic acid.
2.3. Extraction procedures
2.3.1. Raw extracts preparation
For extraction of water-soluble antioxidants and phenolics 0.5 g
of coffee and ginger were poured into 8 mL of boiling water, the
samples were shaken for 30 min at 37 C. After centrifugation
(15 min, 20 C, 8000g), the supernatants were combined, and the
nal volume was brought to 10 ml with distiled water. The nal
extract concentration was 50 mg dry weight (DW)/ml.
2.3.2. In vitro digestion
In vitro digestion was carried out according to the method
described by Gawlik-Dziki (2012) with slight modication. For
simulated gastrointestinal digestion 15 ml of each extract were
mixed with 5 ml of simulated salivary uid (2.38 g Na2HPO4,
0.19 g KH2PO4 and 8 g NaCl), 200 U a-amylase (E.C. 3.2.1.1) in 1 L
H2O, pH 6.75 and shaken for 10 min at 37 C in the absence of light.
Next, the samples were adjusted to pH 1.2 with HCl (5 mM), sus-
pended in 15 mL of simulated gastric uid (300 U/ml of pepsin A,
EC 3.4.23.1 in 0.03 M HCl, pH 1.2) and shaken for 120 min at
37 C in the dark. After simulated gastric digestion, samples were
adjusted to pH 6 with 0.1 M NaHCO3 and suspended in simulated
intestinal juice (0.05 g of pancreatin (activity equivalent 4USP)
and 0.3 g of bile extract in 35 mL 0.1 M NaHCO3), adjusted to pH
7 with 1 M NaOH and nally 5 mL of 120 mM NaCl and 5 mM
KCl were added to the sample. The prepared samples underwent
in vitro intestinal digestion for 120 min in the dark. The nal con-
centration of the resulting gastrointestinally digested extract was
20 mg DM/ml.
2.4. Analytical procedures
Ultra-Performance Liquid Chromatography was used to analyse
compounds of interest using a Waters ACQUITY UPLC system
(Waters Corp., Milford, MA, USA), consisting of a binary pump sys-
tem, sample manager, column manager and PDA detector (also
from Waters Corp.). Waters MassLynx software v.4.1 was used
for acquisition and data processing. The samples were separated
on a BEH C18 column (100 mm  2.1 mm i.d., 1.7 lm, Waters
Corp., Milford, MA, USA), which was maintained at 40 C. The ow
rate was adjusted to 0.40 ml/min. The following solvent system:
mobile phase A (0.1% formic acid in Milli-Q water, v/v) and mobile
phase B (0.1% formic acid in MeCN, v/v) was applied. The gradient
program was as follows: 01.0 min, 5% B; 1.024.0 min, 550% B;
24.025.0 min, 5095% B; 25.027.0 min, 95% B; 27.027.1 min,
955% B; 27.130.0 min, 5% B. Samples were kept at 8 C in the
sample manager. The injection volume of the sample was 2.0 lL
(full loop mode) and samples were analysed in triplicate. Strong
needle wash solution (95:5, methanolwater, v/v) and weak needle
wash solution (5:95, acetonitrilewater, v/v) were used. The detec-
tion wavelength was set at 250 nm at a 5 point/s rate, at 3.6 nm
resolution. The separation was completed in 30 min. Peaks were
assigned on the basis of their UV spectra, mass to charge ratio
(m/z) and ESIMS/MS fragmentation patterns. Chlorogenic acid
 A. Durak et al. / Food Chemistry 166 (2015) 261269
(5-caffeoyl-quinic acid) was used as a group standard for determi-
nation of phenolic compounds.
The MS analyses were carried out on a TQD mass spectrometer
(Waters Corp.) equipped with a Z-spray electrospray interface. The
following instrumental parameters were used for ESIMS analysis
of phenolic compounds (negative ionisation mode): capillary volt-
age, 2.8 kV; cone voltage, 40 V; desolvation gas, N2 800 L/h; cone
gas, N2 100 L/h; source temp. 140 C, desolvation temp. 350 C.
Compounds were analysed in full scan mode (a mass range of
1001600 amu was scanned).
2.5. Determination of ABTS radical scavenging activity
Free radical-scavenging activity was determined by the ABTS+
method according to Re et al. (1999). This reaction is based on dec-
olourization of the green colour of the free ABTS radical cation
(ABTS+). The radical solution was prepared with ABTS and potas-
sium persulfate, diluted in ethanol, to a nal concentration of
2.45 mM and left in the dark for 16 h to allow radical development.
The solution was diluted to reach absorbance measures around
0.700.72 at 734 nm. Then 1.8 mL ABTS+ solution was mixed with
0.04 mL of each sample. The absorbance was measured after
10 min of reaction at 734 nm. Deionized water was used as blank.
Percentage inhibition of the ABTS+ radical was then calculated
using the equation:
scavenging % 1  As=Ac  100
where: As absorbance of sample; Ac absorbance of control (ABTS
solution)
Antiradical activity was expressed as EC50 extract concentra-
tion (mg DW/mL) provided 50% of activity.
All assays were performed in triplicate.
2.6. Determination of ability to lipoxygenase (LOX) inhibition
LOX activity was determined spectrophotometrically at a tem-
perature of 25 C by measuring the increase of absorbance at
234 nm during 1 min (Axelroad, Cheesborough, & Laakso 1981).
The reaction mixture contained 2.45 mL of 1/15 mol/L phosphate
buffer, 0.02 mL of LOX solution (167 U/mL), and 0.05 mL of inhibi-
tor (coffee and cinnamon extract) solution. After preincubation of
the mixture at 30 C for 10 min, the reaction was initiated by add-
ing 0.08 mL 2.5 mM/L linoleic acid. One unit of LOX activity was
dened as an increase in absorbance of 0.001 per minute at
234 nm.
LOX inhibitory activity was expressed as EC50 extract concen-
tration (mg DW/mL) provided 50% of activity.
2.7. Theoretical approach
In accordance with the denition, the half-maximal inhibitory
concentration (IC50) is a measure of the effectiveness of inhibitors.
It is commonly used as a measure of antagonist drug potency in
pharmacological research. The IC50 value is reliable for determining
the activity of a single or two-compound mixture (e.g. isobolo-
graphic analysis) (Williamson, 2001). Further, the EC50 index quan-
titatively measures the amount of extractor extracts mixture that
is required to exhibit half of the measured activity.
The following factor was also determined (Gawlik-Dziki,
2012): The interaction factor (IF), which provides an explanation
for the mode of interaction
IF AM =AT
where, AM = measured activity of a mixture of samples, and
AT = theoretically calculated mixture activity (based on the dose
response of single components at various concentrations).
263
IF value <1 indicates synergistic interaction; IF >1 indicates
antagonism; IF 1 indicates additional interactions.
2.8. Statistical analysis
The experimental results were mean S.D. of three parallel
experiments (n = 9). Statistical analysis was performed using STAT-
ISTICA 7.0 software (StatSoft, Inc., Tulsa, USA) for mean comparison
using Tukeys test at the signicance level a = 0.05.
3. Results
3.1. Qualitative-quantitative analysis of coffee and ginger phenolic
compounds
UPLC/MS analyses allowed the identication of six main groups
of phenolic compounds (primarily phenolic acids) in coffee
extracts (Table 1). The main phenolic compound was 5-caffeoyl
quinic and its derivatives. It should be emphasised that the coffee
extracts contained large amounts of these compounds: 90.80 mg/g
DM before digestion, and 27.80 mg/g DM after a simulated diges-
tion process. Other compounds were from the hydroxycinnamic
acid family, such as feruoyl quinic acid, caffeoyl shikimic acid, fer-
uoyl shikimic acid, dicaffeoyl quinic and caffeoylferuoyl quinic
acid. Concentrations of all phenolic compounds were higher before
in vitro digestion than after this process. Furthermore, extracts of
coffee after digestion did not contain caffeoyl shikimic acid or its
derivatives. It should also be emphasised that coffee extracts con-
tained substantial amounts of caffeine: 71.7 mg/g DM before and
59.05 mg/g DM after digestion in vitro (Table 1). Due to the fact
that chlorogenic acid (5-caffeoylquinic acid) (CGA) was the domi-
nant compound from the hydroxycinnamic acid family in coffee
extracts, it was adopted it as a model compound for coffee.
However, no comprehensive data concerning the phenolic com-
position of ground ginger are available. The current study comple-
ments this gap and shows that ginger can potentially serve as a
rich source of phenolic compounds characterised by a wide-
ranging spectrum of biological activities, including antioxidative
and anti-inammatory potentials. A total of 5 phenolic volatile oils
from ginger infusions have been identied (Fig. 1): four are ginger-
ol derivatives, and the fth identied compound was shogaol.
Moreover, all of these compounds were present in ginger extracts
after digestion. These compounds are water-soluble, but ginger
also contains other phenolic compounds, and one of these is caffeic
acid, the concentration of which in a methanol extract of ginger
was 0.155 mg/g DM (Shan, Cai, Sun, & Corke, 2005). In our work,
in order to determine the interaction between the hydroxycin-
namic acids present in coffee with ginger as an aromatic additive,
it has been assumed on the basis of literature data that the model
compound for ginger is caffeic acid.
Table 1
Concentration of bioactive compounds identied in coffee extract.
Group of phenolic compounds Non-digested Digested
extract (mg/g extract
DW) (mg/g DW)
1. Caffeoyl quinic acid and its 90.80 27.80
derivatives
2. Feruoyl quinic acid and its derivatives 20.40 10.40
3. Caffeoyl shikimic acid and its 15.70
derivatives
4. Feruoyl shikimic acid 4.50 0.60
5. Dicaffeoyl quinic acid and its 14.60 1.10
derivatives
6. Caffeoylferuoyl quinic acid 2.30 0.30
Caffeine 71.7 59.05
264 A. Durak et al. / Food Chemistry 166 (2015) 261269

mg/g DM
Compound Non-digested
Digested extract
extract
1. Rt=17.22 min, [6]-gingediol isomer I 0.3 0.2
2. Rt=17.97 min, [6]-gingediol isomer II 0.3 0.2
3. Rt=18.55 min, [6]-gingerol 3.0 1.4
4. Rt=23.69 min, [8]-gingerol 0.4 0.3
5. Rt=24.36 min, [6]-shogaol 1.4 0.5

Fig. 1. UPLC-MS chromatogram of non-digested (A) and digested (B) ginger extracts.

3.2. Antioxidant activity of single components 3.2.2. LOX-inhibitory activity

For raw extracts, the EC50 values were: 4.85 mg/mL for coffee
3.2.1. Antiradical activity extract and 3.83 mg/mL for ginger extract (Table 2). However, it
In terms of raw extracts, the antiradical potential of coffee was is worth noting that after the simulated digestion process EC50
higher, because, as Table 2 shows, the EC50 value was lower values decreased signicantly to 1.37 mg/mL for coffee and
(1.78 mg/mL) than that for ginger extract (6.40 mg/mL). Further- 1.67 mg/mL for ginger extract (Table 2), which means that the gas-
more, in both samples it was found that simulated gastrointestinal trointestinal system is a good extractor of potentially bioaccessible
digestion caused a signicant increase in antiradical activity; EC50 LOX inhibitors. Model compounds for coffee and ginger were also
values decreased accordingly to 1.49 mg/mL for coffee and LOX-inhibitors: the EC50 value for chlorogenic acid was 41.05 lg/
3.82 mg/mL for ginger (Table 2). mL, and that for caffeic acid was 22.86 lg/mL (Table 2). Thus, caf-
Caffeic acid alone, as a pure chemical compound, demonstrated feic acid was a more potent inhibitor of LOX than chlorogenic acid.
ABTS radical scavenging activity and the EC50 value was 22.37 lg/
mL. On the other hand, chlorogenic acid, as a model compound for 3.3. Interaction assay
coffee extract, had a lower antioxidant capacity, and the EC50 value
was 36.76 lg/mL (Table 2). The antioxidant capacity of the water-soluble compounds of
coffee extract in combination with ginger extract was revealed
using the ABTS method. As shown in Fig. 2A, the isobole adopted
Table 2
a concave form and this result indicates that antiradical scavengers
Comparison of EC50 values for coffee and ginger extracts and pure chemical included in coffee and ginger acted synergistically before in vitro
compounds assumed as model compounds. digestion. Interesting results were obtained during the isobolo-
Sample Extracts Activity EC50 (mg/mL)
graphic analysis of the extracts subjected to the simulated diges-
tion. The changes that occurred in the extracts of coffee and
Coffee Raw Antiradical potential 1.78
Ginger 6.40
ginger changed their ability to scavenge ABTS radicals, which can
Coffee LOXI activity 4.85 be concluded because the isobole curve has a slightly convex
Ginger 3.83 shape. This means that after the process of digestion in vitro an
Coffee Digested Antiradical potential 1.49 additive reaction was observed (Fig. 2B). Furthermore, in the case
Ginger 3.82 of pure chemical compounds, an antagonism was found (Fig. 2C).
Coffee LOXI activity 1.37 On examining the ability to inhibit LOX activity, an antagonistic
Ginger 1.67
interaction between coffee and ginger non-digested extracts was
Pure chemical standards EC50 (lg/mL) observed. As Fig. 3A shows, the isobole curve is said to be convex.
Chlorogenic acid Antiradical potential 36.76
Furthermore, it was found that, after the process of digestion,
Caffeic acid 22.37
in vitro extracts acted synergistically (Fig. 3B). Consequently, the
Chlorogenic acid LOXI activity 41.05
changes that occurred during the digestion process caused a
Caffeic acid 22.86
change in the type of interaction between the test extracts. Fur-
 A. Durak et al. / Food Chemistry 166 (2015) 261269 265

Fig. 2. Isobole curves for the 50% ABTS radical scavenging activity of coffee and ginger mixtures (A) before digestion, (B) after digestion in vitro and mixtures of pure
chemical compounds (C).

thermore, the same kind of interaction was observed for pure
chemical compounds, where the synergism seemed to be stronger
(Fig. 3C).
The isobolographic analysis is quite time-consuming and com-
plicated, that is why an interaction factor (IF) has been used which
provides an explanation for the mode of interaction. It is a simple
way to make a preliminary assessment of the type of interactions
between the examined extracts or chemical compounds. As Table 3
presents, isobole curves shown in Figs. 2 and 3 are conrmed by IF,
as calculated using the ratios of the measured activity of samples
and theoretically calculated mixture activity (based on the dose
response of single components at various concentrations), and
expressed as EC50.
4. Discussion
The proven health benets of coffee brews amply justify the
inclusion of this infusion as a functional food. Bisht and Sisodia
266 A. Durak et al. / Food Chemistry 166 (2015) 261269

Fig. 3. Isobole curves for the 50% LOX-inhibitory activity of coffee and ginger mixtures (A) before digestion, (B) after digestion in vitro and mixtures of pure chemical
compounds (C).

(2010) recently noted that worldwide coffee is the most frequently
consumed functional food. This global nature of coffee drinking
means that it impacts upon a broader demographic population
than other functional foods that act on a more dened population
(Drea & da Costa, 2005). New benecial properties of the coffee
beverage are constantly being discovered (Esquivel & Jimnez,
2012). A coffee brew contains many of the most important known
functional ingredients, such as avonoids (catechins and anthocy-
anins), and caffeic and ferulic acids (Meletis, 2006). In addition,
other biologically active compounds found in coffee include
nicotinic acid, trigonelline, quinolinic acid, tannic acid, pyrogallic
acid and caffeine (Minamisawa, Yoshida, & Takai, 2004). The
beverage is also known for the antioxidant properties of its compo-
nents caffeine, CGA, hydroxycinnamic acids and melanoidins
 A. Durak et al. / Food Chemistry 166 (2015) 261269
Table 3
Comparison of interaction factors (IF) of mixtures of coffee with ginger and pure
chemical compounds taken as model for experimental material.
Sample Extracts Activity AM* AT** IF***
Coffee/ginger mixture Raw Antiradical 2.61 4.09 0.64
(1:1) potential
LOXI activity 5.38 4.21 1.28
Digested Antiradical 2.86 2.66 1.08
potential
LOXI activity 1.01 1.52 0.66
Chlorogenic acid Antiradical 59.66 47.95 1.24
/caffeic acid potential
mixture (1:1) LOXI activity 20.00 31.96 0.63
* reactive oxygen species and inhibit the peroxidation of lipids. Thus,
Measured activity (AM) of a mixture of samples (expressed as EC50 (mg/mL)).
**
Theoretical calculated activity (AT) (based on the dose response of single com-
ponents at various concentrations) (expressed as EC50 (mg/mL)).
***
Interaction factor (IF) value <1 indicates synergistic interaction; IF > 1 indicates
antagonism; IF  1 indicates additional interactions.
(Run-Henares & Morales, 2007; Vignoli, Bassoli, & Benassi,
2011). Antioxidants of the hydroxycinnamic acid group, such as
combined or conjugated forms of caffeic, chlorogenic, coumaric,
ferulic and sinapic acids, are also found in coffee beverages
(Manach, Scalbert, Morand, Rmsy, & Jimnez, 2004). In the cur-
rent study, most of the coffee compounds, which are listed above
on the basis of literature data, have been identied. Moreover, only
caffeoyl shikimic acid and its derivatives were not present in the
extract after the digestion process. This means that other groups
of phenolic compounds are able to survive the processes taking
place during digestion, and potentially have a positive impact on
the body. Bioaccessibility is dened as the amount of a compound
released from the solid food matrix, and thereby capable of passing
through the intestinal barrier. However, the proportion of the com-
pound that is digested, absorbed, and metabolized through the
normal pathways, referred to as its bioavailability, is far more
important for the nutraceutical potential of the food (DArchivio,
Filesi, Vari, Scazzocchio & Masella, 2010). In turn, Saura-Calixto,
Serrano, and Goi (2007) in their work stated that polyphenols
from beverages are completely bioaccessible because they pass
directly into the intestinal uids, and they are the largest contrib-
utors of bioaccessible polyphenols in the small intestine. In order
to determine the potential bioaccessibility of compounds bearing
antioxidative potential, the antioxidative properties of raw coffee
and ginger extracts (before digestion in vitro) have been analysed
and also those extracts subjected to simulated gastrointestinal pro-
cessing. This provides a rationale for the relevance of this work, as
not all initial polyphenols will actually play a role in the health sta-
tus of a potential tentative consumer. It was observed that the con-
centration of individual bioactive compounds decreased after the
process of digestion, and the properties of extracts after simulated
digestion were different from those of the raw extracts of coffee
and ginger. It is worth noting that active compounds were
extracted with hot water in order to determine the amount of poly-
phenols present in coffee and ginger infusions as prepared by
consumers.
In the present work, the respective antioxidant activities of cof-
fee and ginger extracts were determined with an ABTS assay. As
Table 2 shows, antioxidant activity was observed for coffee and
ginger infusions. Neutralization of free radicals is an antioxidant
defence mechanism, which is found in medicinal plants and food
ingredients. In vitro studies on the ability of the compounds con-
tained in coffee extracts to neutralize free radicals have shown that
ferulic acid, caffeic acid and lastly chlorogenic acid (CGA) are
mainly responsible for this effect (Gomez-Ruiz et al., 2007).
Record and Lane (1996) proved that the content of phenolic com-
pounds increased after acid hydrolysis of tea infusions (conditions
similar to those prevailing in the human gastrointestinal tract). A
267
decrease in the concentration of phenolic compounds was noted
with the time of incubation at pH 7.5 (conditions such as exist in
the small intestine). The current study showed a decrease in the
quantity and quality of phenolic compounds in both the coffee
and ginger extracts. In turn, as Gawlik-Dziki (2004) reported that
phenolic acid levels may increase upon heating in acidic conditions
as a result of the hydrolysis of ester bonds and glycosidic hydroly-
sable tannins (as is the case during digestion in vitro). In the exper-
iment the digestion process was too short-lived to lead to the
complete hydrolysis of tannins present in the extract of coffee.
Furthermore, the antioxidant properties of ginger extracts are
attributable to the ability of its phenolic constituents to quench
they could prevent or decrease the damage in a human body
caused by free radicals, which, according to Aruoma et al. (1997)
and Valko, Izakovic, Mazur, Rhodes, and Telser (2004), attack bio-
logical macromolecules such as lipids, proteins and DNA. By
averting the oxidation of the phospholipids of cell membranes,
specic cell permeability is preserved and the cell metabolism is
undisturbed.
The constituents responsible for the taste and properties of gin-
ger are a homologous series of phenolic ketones, known as [4]-,
[6]-, [8]-, [10]- and [12]-gingerols [2]. The shogaol series of com-
pounds, even more pungent than the gingerols, is virtually absent
from fresh ginger, and is derived from the corresponding gingerols
during thermal processing or long-term storage (He, Bernart, Lian,
& Lin, 1998). In the current study, ginger was used as a ground
spice, and the compounds that were identied in the aqueous
extract were: [6]-gingerol, [8]-gingerol, [6]-gingediol isomer I,
[6]-gingediol isomer II and [6]-shogaol, as Fig. 1 shows. The repre-
sentative of phenolic acids in ginger is caffeic acid. Its concentra-
tion in the methanol extract was recorded at the level of
0.155 mg/g DM (Shan et al., 2005). He et al. (1998) found that gin-
ger had a good antioxidant effect, probably due to the presence of
tocopherols, phospholipids, and phenolic compounds, which have
aromatic rings, or due to the synergistic effect of these compounds
(Kawamura, Okada, Futami, & Fukuba, 1993).
In the present work, the antioxidant potential of coffee and gin-
ger extracts was also determined as the ability to LOX inhibition. In
the recent literature, there is a lack of information concerning the
LOX-inhibitory ability of coffee extracts and their bioaccessibility.
Therefore, an attempt was made to study the LOX-inhibitory activ-
ities of this commonly consumed beverage. It should be mentioned
that the studied extracts of coffee and ginger were good sources of
potentially bioaccessible LOX inhibitors, which are released during
gastrointestinal digestion. A comparison of EC50 values shows that
pH digestion had a signicant effect on LOX inhibition. The tested
extracts effectively prevented lipids from oxidising and it is inter-
esting that varying pH conditions (simulating those occurring in
the gastrointestinal system) caused a signicant enhancement of
the activity of both extracts. Water extract possessed lower LOX
inhibitory activity before digestion, whereas after digestion its
EC50 decreased to 1.37 mg/mL for coffee and 1.67 mg/mL for ginger
extract, respectively.
Synergistic interactions are of vital importance in phytomedi-
cines to explain the difculties in always isolating a single active
ingredient, and they explain the efcacy of apparently low doses
of active constituents in a herbal product. This concept, that a
whole or partially puried extract of a plant offers advantages over
a single isolated ingredient, also underpins the philosophy of her-
bal medicine (Williamson, 2001). Unlike in the case of synthetic
pharmaceuticals based on the activity of single (chemical) active
compounds, numerous phytochemicals act in a benecial manner
via an addition of synergistic activity in target sites connected to
physiological processes. This idea has found an application in
pharmacology during investigations into combinations of a few
268 A. Durak et al. / Food Chemistry 166 (2015) 261269
metabolites in multidirectional therapy (Birskin, 2000). The
method usually used for the identication of interactions between
active compounds is isobolographic analysis. This method is inde-
pendent of the activity of a mechanism; however, it should be
emphasised that this analysis is quite complicated and labour-
intensive. The issues concerning the application of isobolographic
analysis for the determination of the interactions between the
anti-inammatory compounds (LOX inhibitors) of owers of lime
and common dandelion were undertaken in a study by Gawlik-
Dziki (2011).
In this study it was observed that the isobole curves for the 50%
ABTS radical scavenging activity of coffee and ginger mixtures
before digestion have a concave shape (Fig. 2A). This means that
compounds present in the tested extracts demonstrate a synergis-
tic effect. It should be noted, however, that the shape of the isobole
curve for coffee and ginger mixtures after digestion in vitro has a
different shape, which shows that the changes in the extracts as
a result of the digestion process mean that an additive reaction is
observed between active compounds. In turn, for pure chemical
standards, a convex isobole was found (Fig. 2C), meaning that these
compounds acted antagonistically.
The isobolographic analysis was also performed for mixtures of
coffee with ginger in the context of their ability to inhibit LOX
activity. As Fig. 3A shows, active compounds of both extracts acted
antagonistically before digestion. It is worth noting that after
in vitro digestion the isobole is concave (Fig. 3B), and a synergistic
interaction between the studied extracts has been demonstrated.
In the case of chemical standards, a synergistic interaction was also
observed, indicated by the concave shape adopted by the isobole
(Fig. 3C). Moreover, most studies on polyphenol bioavailability
mainly use pure single compounds (isolated from food or chemi-
cally synthesised), although their bioavailability from whole foods
might be substantially different (Saura-Calixto et al., 2007). Conse-
quently, the desire was to compare types of interactions between
whole extracts of coffee and ginger with respect to pure chemical
compounds that were used as model compounds for those com-
pounds present in the tested material.
A denitively less complicated method for the evaluation of the
interactions between bioactive compounds is the determination of
the interaction factor (IF) proposed by Gawlik-Dziki (2012). This
proposed way of studying interactions, as in isobolographic analy-
sis, is independent of the mechanism of active compound activity
and requires a linear relationship between an activity and sample
concentration. Its crucial advantage is the possibility of conducting
studies of interactions between any number of components and
the fact that it is markedly less complicated. Moreover, the
strength of interaction may be approximately estimated based
on the IF value. In the current work it has been shown that the type
of interaction determined on the basis of isobolographic analysis
(Figs. 2 and 3) is conrmed by IF (Table 3). Therefore, as mentioned
above, this index can be used in the initial assessment of the inter-
action between the two active ingredients.
5. Conclusion
The obtained results suggest that processes occurring in the
gastrointestinal tract might improve the antioxidant and anti-
inammatory properties of coffee and ginger extracts. However,
the interaction with the food matrix and/or quantitative and qual-
itative changes in the content of phenolic compounds led to signif-
icant differences in the interactions between phenolic compounds
present in the tested extracts. Taking into account the analysis of
the interaction between the pure compounds, the type of interac-
tion was similar to that recorded with extracts after digestion. In
turn, raw extracts interact with each other quite differently than
pure chemical compounds.
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