Food Chemistry 162 (2014) 81–88

Contents lists available at ScienceDirect

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

Coffee with cinnamon – Impact of phytochemicals interactions

on antioxidant and anti-inflammatory in vitro activity
Agata Durak a,⇑, Urszula Gawlik-Dziki a, Łukasz Pecio 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 paper evaluates the potential bioaccessibility and interactions between antiradical and anti-
Received 31 October 2013 inflammatory compounds from coffee and cinnamon. Results obtained for whole plant material extracts
Received in revised form 4 March 2014 were compared with those for chlorogenic and cinnamic acids (the main bioactive constituents of the
Accepted 8 March 2014
study material). All samples, coffee, cinnamon and a mixture of the two showed abilities to scavenge free
Available online 16 April 2014
radicals and to inhibit lipoxygenase (LOX) activity. Both activities increased after simulated gastrointes-
tinal digestion. In the mixture antiradical phytochemicals acted antagonistically – isoboles adopted the
Keywords:
convex form. The same interactions were determined for chemical standards. The water-extractable
Coffee
Cinnamon
LOX inhibitors acted synergistically – the isobole curve was ‘‘concave’’. The same type of interaction
Interactions was determined for standard compounds. Interestingly, after digestion in vitro a slight antagonism in
Isobolographic analysis the action of LOX inhibitors was observed. The results show that the food matrix and/or its changes
Interaction factor during digestion may play an important role in creating the biological properties.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction and radical scavenging activities) such as chlorogenic acid, nico-

Oxidative stress is a biological state that occurs when a cell’s
antioxidant capacity is overwhelmed by reactive oxygen species
(ROS), causing a redox imbalance. As such, ROS and oxidative stress
as a whole have been suggested to participate in the initiation and/
or propagation of chronic diseases such as cardiovascular and
inflammatory diseases, cancer, and diabetes (Valko et al., 2007).
ROS generation during inflammation is bound, inter alia,
with lipoxygenase (LOX) activity (Gawlik-Dziki et al., 2013). This
enzyme catalyses the oxygenation of polyunsaturated fatty acids
containing a cis,-cis-1,4-pentadiene system to hydroperoxides.
The lipoxygenase pathway of the arachidonic acid metabolism pro-
duces ROS and these reactive forms of oxygen and other arachi-
donic acid metabolites might play a role in inflammation and
tumour promotion. Inhibition of the arachidonic acid metabolism
prevents tumour promotion in animal models (Juntachote &
Berghofer, 2005).
Despite the controversial effects of coffee on human health
(Taylor & Demmig-Adams, 2007), coffee is a commonly consumed
beverage in Europe and America (Debry, 1994). The coffee bever-
age is rich in biologically active substances (possessing antioxidant
⇑ Corresponding author. Tel.: +48 81 4623329; fax: +48 81 4623324.
E-mail address: agatadurak@gmail.com (A. Durak).
tinic acid, trigoelline, quinolic acid, tannic acid, pyrogallic acid
and, of course, caffeine (Cammerer & Kroh, 2006; Dórea & da
Costa, 2005). Spices and herbs have been added to foods since
ancient times, not only as flavouring agents, but also as folk med-
icines and food preservatives. Presently, there is increasing interest
in spices and aromatic herbs, both in industry and in scientific
research, because of their strong antioxidant properties (Deepa,
Ayesha, Nishtha, & Thankamani, 2013). It is worth noting that
many herbs and spices usually used to flavour dishes contain phe-
nolic compounds which have been reported to show good antiox-
idant activity (Zheng & Wang, 2001). The cinnamon used in this
study as a coffee supplement affects not only the flavour and
aroma of the beverage, but also its antioxidant properties, because
this spice is a source of bioactive compounds such as cinnamic acid
and coumarin (Shan, Cai, Sun, & Corke, 2005). Furthermore, cinna-
mon is a popular flavouring ingredient, widely used in food prod-
ucts. It has exhibited health beneficial properties, such as
antimicrobial activity, for controlling glucose intolerance and dia-
betes, inhibiting the proliferation of various cancer cell lines, and
treating the common cold (Murcia et al., 2004). Cinnamon extracts
exhibit a protective capacity against irradiation induced lipid per-
oxidation in liposomes, and quench hydroxyl radicals and hydro-
gen peroxide (Peschel et al., 2006). The antioxidant properties of
cinnamon extracts are attributable to the ability of their phenolic

http://dx.doi.org/10.1016/j.foodchem.2014.03.132
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
82 A. Durak et al. / Food Chemistry 162 (2014) 81–88
constituents to quench reactive oxygen species (Moselhy & Junbi,
2010).
To exert their biological properties, polyphenols have to be
available to some extent in the target tissue. Therefore, the
biological properties of dietary polyphenols may depend on their
absorption in the gut and their bioavailability. The amount of bio-
accessible food polyphenols may differ quantitatively and qualita-
tively from polyphenols included in food databases. Moreover,
most studies on polyphenol bioavailability mainly use pure single
molecules (isolated from food or chemically synthesised), even
though their bioavailability from whole foods may be substantially
different (Saura-Calixto, Serrano, & Goñi, 2007). Furthermore, the
activity of phenolic compounds studied in vitro (after their isola-
tion from the food matrix) does not have to be in accordance with
the activity demonstrated in the human organism. In vitro models
based on human physiology are simple, cheap and repeatable tools
for the study of food component bioaccessibility. They are widely
used for the study of structural changes, digestibility and food
component release in simulated conditions of the alimentary tract
(Oomen et al., 2002).
In contrast to synthetic pharmaceuticals, based upon single
chemicals, many phytomedicines exert their beneficial effects
through the additive or synergistic action of several chemical com-
pounds acting at a single or multiple target sites associated with a
physiological process. The method used for identification of inter-
actions between active compounds is isobolographic analysis
(Williamson, 2001). This method is independent of the activity
mechanism; however, it should be emphasised that this analysis
is quite complicated and labour-consuming. A definitely less com-
plicated method for the determination of interactions between
mixtures of components – interaction factor (IF) – was proposed
by Gawlik-Dziki (2012a). This approach to the study of interac-
tions, as with isobolographic analysis, is independent of the mech-
anism of activity but requires a linear relationship between an
activity and a sample concentration. Moreover, the ‘‘strength’’ of
interaction may be estimated approximately from the IF value.
Thus, the aim of this study was to test two hypotheses: (1)
interactions between bioactive phenolics play a crucial role in
the creation of the nutraceutical potential of coffee supplemented
with cinnamon, (2) interaction with the food matrix and/or
changes during simulated gastrointestinal digestion cause signifi-
cant differences in relationships between the main phenolic com-
pounds 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 Sigma–Aldrich company. Acetonitrile and
methanol gradient HPLC grade and formic acid LC-MS grade for
LC-UV-MS separations were purchased from J.T. Baker (Phillips-
burg, NJ). Water was purified in-house with a Milli-Q water purifi-
cation system Simplicity-185 (Millipore Co.). 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. Cinnamon was
bought from a local market (Lublin, Poland) in the form of ground
spice.
The coffee brew and the cinnamon 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/cinnamon.
Model compound solutions were prepared in water and the
final concentration was 10 lg/mL. The chlorogenic acid solution
and the solution of cinnamic acid were analysed separately
and in appropriate combinations (4:1; 3:2; 1:1; 2:3; 1:4 v/v)
chlorogenic acid/cinnamic acid.
2.3. Raw extract preparation
For extraction of water-soluble antioxidants and phenolics 8 mL
of boiling water were poured over 0.5 g of coffee and cinnamon,
the samples were shaken for 30 min at 37 °C. After centrifugation
(15 min, 20 °C, 8000g), the supernatants were decanted from the
precipitate, and the final volume was brought to 10 mL with dis-
tilled water. The final extract concentration was 50 mg dry weight
(DW)/mL.
2.4. In vitro digestion
In vitro digestion was carried out according to the method
described by Gawlik-Dziki (2012a) with slight modification. For
simulated gastrointestinal digestion 15 mL of each extract were
mixed with 5 mL of simulated salivary fluid (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 darkness. Next,
the samples were adjusted to pH 1.2 with HCl (5 mM), suspended
in 15 mL of simulated gastric fluid (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 darkness. 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 4  USP)
and 0.3 g of bile extract in 35 mL 0.1 M NaHCO3), adjusted to pH
7 with 1 M NaOH and finally 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 darkness. The final con-
centration of the resulting gastrointestinally digested extract was
20 mg DM/mL.
2.5. Ultra-performance liquid chromatography
Compounds of interest were analysed using a Waters ACQUITY
UPLC™ system (Waters Corp., Milford, MA, USA), consisting of a
binary pump system, 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 flow 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: 0–1.0 min, 5% B;
1.0–24.0 min, 5–50% B; 24.0–25.0 min, 50–95% B; 25.0–27.0 min,
95% B; 27.0–27.1 min, 95–5% B; 27.1–30.0 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, methanol–water,
v/v) and weak needle wash solution (5:95, acetonitrile–water,
v/v) were used. The detection 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 ESI-MS/MS fragmentation patterns.
Chlorogenic acid (5-caffeoyl-quinic acid) was used as a group stan-
dard for determination of phenolic compounds.
 A. Durak et al. / Food Chemistry 162 (2014) 81–88
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 ESI-MS 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 (mass range of
100–1600 amu was scanned).
2.6. 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
decolourisation 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, at final concentration of
2.45 mM and left at dark for 16 h to allow radical development.
The solution was diluted to reach absorbance measures around
0.70–0.72 at 734 nm. 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. Deionised water was used as blank.
Percentage inhibition of the ABTS+ radical was then calculated
using the equation:
Scavenging % ¼ ½1  ðAs =Ac Þ  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.
2.7. 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 over a 1 min period (Axelroad, Cheesborough, & Laakso,
1981). The reaction mixture contained 2.45 mL of 1/15 mol/L phos-
phate buffer, 0.02 mL of LOX solution (167 U/mL), and 0.05 mL of
inhibitor (coffee and cinnamon extract) solution. After preincuba-
tion of the mixture at 30 °C for 10 min, the reaction was initiated
by adding 0.08 mL 2.5 mM/L linoleic acid. One unit of LOX activity
was defined 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.
2.8. Theoretical approach
In accordance with the definition, 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 extract mixture that is
required to exhibit half of the measured activity.
The following factor was also determined (Gawlik-Dziki,
2012a):
- The interaction factor (IF)1, which provides an explanation for
the mode of interaction
1
It must be kept in mind that the proposed formula is correct only in cases when
the measure activity measure of activity is a value inversely related to the activity, e.g.
IC50 (the higher the activity, the lower the IC50). If activity is expressed as Trolox
equivalents, the formula must be reversed).
83
IF ¼ AM =AT
where AM = measured activity of a mixture of samples, and AT = the-
oretically calculated mixture activity (based on the dose response of
single components at various concentrations).
IF value <1 indicates synergistic interaction; IF > 1 indicates
antagonism; IF  1 indicates additional interactions.
2.9. Statistical analysis
The experimental results were mean ± S.D. of three parallel
experiments (n = 9). Statistical analysis was performed using
STATISTICA 7.0 for mean comparison using Tukey’s test at the
significance level a = 0.05.
3. Results
3.1. Qualitative–quantitative analysis of coffee and cinnamon phenolic
compounds 165
UPLC/MS analyses allowed the identification of 14 phenolic
compounds (primarily phenolic acids) in coffee extracts (Fig. 1).
The main phenolic acids were compounds from the hydroxycin-
namic acid family, such as caffeoyl quinic acid and its isomers, fer-
uoyl quinic acid, caffeoyl shikimic acid, dicaffeoyl quinic acid and
caffeoyl-feruoyl quinic acid. The concentrations of all phenolic
compounds were higher before in vitro digestion than after this
process. Furthermore, the extract of coffee after digestion did not
contain the following compounds: caffeoyl shikimic acid I, caffeoyl
shikimic acid II, and clovamide; and the concentrations of other
phenolic compounds were considerably lower than in non-
digested extracts. It should also be emphasised that coffee extracts
contained large amounts of caffeine: 71.7 mg/g before and
59.0 mg/g after digestion in vitro (Fig. 1). Furthermore, we adopted
chlorogenic acid as a model for the coffee extract. Our coffee sam-
ples contained 30.30 mg/g of 5-caffeoyl-quinic acid in the non-
digested extract and the content of this compound decreased to
12.90 mg/g after the digestion process (Fig. 1).
However, no comprehensive data concerning the phenolic
composition of cinnamon are available. Our study fills this gap
and shows that cinnamon can potentially serve as a rich source
of phenolic compounds characterised by a wide-range spectrum
of biological activities, including antioxidative potential. We have
identified 8 phenolic compounds from cinnamon infusions
(Fig. 2): four compounds from the proanthocyanidin class of
flavonoids, cinnamic acid as a representative of the group of
hydroxycinnamic acids and other polyphenols such as: coumarin,
(Z)-cinnamaldehyde, (E)-cinnamaldehyde. Moreover, all of these
compounds were present in the cinnamon extract after digestion.
In our study, we have assumed that the model compound for cin-
namon is cinnamic acid.
3.2. Antioxidant activity of single components
3.2.1. Antiradical activity
Taking into account raw extracts, a similar antiradical power
was observed for both samples: EC50 = 1.86 mg/mL for the coffee
extract and EC50 = 1.52 mg/mL for the cinnamon sample (Table 1).
Furthermore, in both samples it was found that simulated gastroin-
testinal digestion caused a significant increase in antiradical activ-
ity; EC50 values decreased accordingly to 1.47 mg/mL for coffee and
to 1.18 mg/mL for cinnamon (Table 1).
Cinnamic acid alone, as a pure chemical compound, demon-
strated ABTS radical scavenging activity and the EC50 value was
119.70 lg/mL. On the other hand, chlorogenic acid, as a model
84 A. Durak et al. / Food Chemistry 162 (2014) 81–88

[mg/g DW]
Non-
Compound Digested
digested
extract
extract
1. Rt=2.51 min, caffeoyl quinic acid isomer 19.8 7.10
2. Rt=3.75 min, 3-caffeoyl quinic acid 40.7 7.80
3. Rt=3.98 min, caffeine 71.7 59.00
4. Rt=4.18 min, 5-caffeoyl quinic acid 30.3 12.90
5. Rt=5.83 min, feruoyl quinic acid I 12.3 5.40
6. Rt=5.94 min, feruoyl quinic acid II 8.10 5.00
7. Rt=6.09 min, caffeoyl shikimic acid I 9.40 -
8. Rt=6.56 min, caffeoyl shikimic acid II 6.30 -
9. Rt=8.18 min, feruoyl shikimic acid 4.50 0.60
10. Rt=8.52 min, dicaffeoyl quinic acid I 6.60 0.40
11. Rt=8.61 min, dicaffeoyl quinic acid II 3.80 0.30
12. Rt=9.43 min, dicaffeoyl quinic acid III 4.20 0.40
13. Rt=10.14 min, caffeoyl-feruoyl quinic acid 2.30 0.30
14. Rt=11.52 min, clovamide 4.90 -
A

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

compound for coffee extract had a much higher antioxidant capac-
ity, and the EC50 value was 37.08 lg/mL (Table 1).
3.2.2. LOX-inhibitory activity
With reference to raw extracts, the EC50 values were similar:
4.22 mg/mL for the coffee extract and 4.98 mg/mL for the cinna-
mon extract (Table 1). However, it is worth noting that after the
simulated digestion process EC50 values decreased significantly to
1.45 mg/mL for coffee and 1.37 mg/mL for the cinnamon extract
(Table 1), which means that the gastrointestinal system is a good
extractor of potentially bioaccessible LOX inhibitors. Model com-
pounds for coffee and cinnamon were also LOX-inhibitors: the
EC50 value for chlorogenic acid was 39.50 lg/mL, and for cinnamic
acid it was 45.38 lg/mL (Table 1). Thus, a similar correlation was
observed here as in the case of extracts. Chlorogenic acid was a
more potent inhibitor of LOX than cinnamic acid.
3.3. Interaction assay
The antioxidant capacity of the water-soluble compounds of
coffee extract in combination with cinnamon extract has been
revealed by the ABTS method. As being present in Fig. 3(A and
B), isoboles took the convex form. This result indicates that anti-
radical scavengers included in coffee and cinnamon acted as antag-
onists both prior to digestion and after this process. Furthermore,
in the case of pure chemical compounds, we observed the same
kind of interaction (Fig. 3C).
In examining the ability to inhibit LOX activity, we observed a
synergistic interaction between coffee and cinnamon extract. As
Fig. 4A shows, isobole has a concave shape. Furthermore, we
observed the same kind of interaction for pure chemical com-
pounds, and the synergism seems to be stronger (Fig. 4C). Interest-
ing results were obtained during the isobolographic analysis of
tested extracts subjected to simulated digestion. The changes that
have occurred in the extracts of coffee and cinnamon significantly
increased their ability to inhibit LOX activity, but after the estab-
lishment of appropriate mixtures of these extracts (as discussed
above), we found that after the process of digestion in vitro extracts
act slightly antagonistically (Fig. 4B).
Isobolographic analysis is quite time-consuming and compli-
cated, that is why we use the interaction factor (IF), 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 2 shows,
isobole curves shown in Figs. 3 and 4 are confirmed by IF, as calcu-
lated by the ratios of the measured activity of samples and the
 A. Durak et al. / Food Chemistry 162 (2014) 81–88 85

[mg/g DW]
Non-
Compound Digested
digested
extract
extract
1. Rt=4.29 min, procyanidin tetramer 5.0 0.9
2. Rt=4.80 min, procyanidin dimer 2.2 0.3
3. Rt=4.99 min, procyanidin trimer I 7.5 4.3
4. Rt=5.45 min, procyanidin trimer I 4.0 1.3
5. Rt=8.52 min, coumarin 4.8 4.7
6. Rt=9.03 min, cinnamic acid 1.0 0.8
7. Rt=11.62 min, (Z) cinnamaldehyde 4.5 4.1
8. Rt=11.94 min, (E) cinnamaldehyde 35.7 26.2 B

Fig. 2. UPLC-MS chromatogram of non-digested (A) and digested (B) cinnamon extracts.

Table 1
Comparison of EC50 values for antiradical and LOX-inhibitory activity.

Sample Extracts Activity EC50 [mg/mL] Curve equation to calculate the EC50 values R2
Coffee Raw Antiradical potential 1.86 y = 26.067x + 1.46 0.987
Cinnamon 1.52 y = 34.395x  2.29 0.985
Coffee LOXI activity 4.22 y = 12.096x  1.05 0.991
Cinnamon 4.98 y = 10.273x  1.11 0.996
Coffee Digested Antiradical potential 1.47 y = 33.624x + 0.67 0.996
Cinnamon 1.18 y = 37.983x + 5.13 0.978
Coffee LOXI activity 1.45 y = 32.263x + 3.22 0.990
Cinnamon 1.70 y = 35.54 + 1.15 0.973
Chlorogenic acid Antiradical potential 0.007 y = 1.173x + 6.53 0.988
Cinnamic acid 0.119 y = 0.405x + 1.54 0.984
Chlorogenic acid LOXI activity 0.039 y = 1.143x + 4.87 0.995
Cinnamic acid 0.045 y = 1.0916x + 0.46 0.997

theoretically calculated mixture activity (based on the dose
response of single components at various concentrations) and
expressed as EC50.
4. Discussion
Plant foods and beverages are the main sources of antioxidants
in the diet. Dietary antioxidants are complex mixtures of hundreds
of compounds and the gastrointestinal tract is the major site of
their synergistic action. Plant food contains a complex array of
phenolic compounds that may help to increase the antioxidant
capacity of foods (Saura-Calixto et al., 2007). Phenolic substances
have been shown to be responsible for the antioxidant activity of
plant materials (Hinneburg, Dorman, & Hiltunen, 2006). The poly-
phenols were extracted with hot water in order to determine the
amount of polyphenols present in coffee and cinnamon infusions
as prepared by consumers.
In the present study, the respective antioxidant activities of cof-
fee and cinnamon extracts were determined with an ABTS assay.
As Table 1 shows, antioxidant activity was observed for coffee
and cinnamon infusions. Neutralisation of free radicals is an anti-
oxidant defense mechanism, which has medicinal plant and food
ingredients. In vitro studies into the ability of the compounds con-
tained in the coffee extract to neutralise free radicals have shown
that this effect is mainly due to the activity of ferulic acid, caffeic
acid and then chlorogenic acid (CGA) (Gomez-Ruiz, Leake, &
86 A. Durak et al. / Food Chemistry 162 (2014) 81–88

2.50 Furthermore, the antioxidant properties of cinnamon extracts

A are attributable to the ability of their phenolic constituents to
2.00
Coffee [mg/mL]

quench reactive oxygen species. Cinnamaldehyde and cinnamic

1.50
acid are major phenolic components of cinnamon extracts (Cheng
1.00 et al., 2012). Moreover, the results obtained by Mathew and
0.50 Abraham (2006) have shown that the methanolic extract of cinna-
mon contains a number of antioxidant compounds which can
0.00
effectively scavenge reactive oxygen species including superoxide
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
Cinnamon [mg/mL]
anions and hydroxyl radicals as well as other free radicals under
in vitro conditions. Moreover, the hydrogen donating ability of all
the experimental compounds and the extract has been proven
1.6
B through the assessment of reducing power and DPPH scavenging
1.4
activity. Even though cinnamon extracts are weak chelators of
1.2
Coffee [mg/mL]

metal ions, their free radical scavenging capacity is comparable

1
to that of synthetic antioxidants such as BHA. Cinnamon
0.8
extracts possess antioxidant properties, which are concentration-
0.6
dependent.
0.4
In the present study, the antioxidant potential of coffee and
0.2 cinnamon extracts was also determined as the ability to LOX inhi-
0 bition. The interaction of flavonoids with mammalian 15-lipoxyge-
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40
nase-1 merits particular attention, as this enzyme is a potential
Cinnamon [mg/mL]
target for the health-preserving effect of flavonoids (Sadik, Sies, &
70.00 Schewe, 2003). In the recent literature there is a lack of informa-
Chlorogenic acid [µg/mL]

60.00 C tion concerning the LOX-inhibitory ability of coffee extracts and

50.00
40.00
30.00
20.00
10.00
0.00
0.00 20.00 40.00 60.00 80.00
Cinnamic acid [µg/mL]
Fig. 3. Isobole curves for 50% ABTS radical scavenging activity of coffee and
cinnamon mixtures before digestion (A), after digestion in vitro (B) and mixtures of
standard compounds (C).
Ames, 2007). The studies of this group of researchers suggest that
two of the most important colonic metabolites of CGA – m-couma-
ric and 3-(hydroxyphenyl) propionic acid – have a high antioxidant
activity. Both compounds showed antioxidant values only slightly
lower than that of chlorogenic acid. Furthermore, it has been well
established that CGA derivatives are the predominant antioxidants
in coffee brews. Chlorogenic acids are a family of esters formed
between trans-cinnamic acids and quinic acid. The most usual
and widespread individual chlorogenic acid is formed between caf-
feic acid and quinic acid and the most abundant CGAs in coffee are
caffeic acid, including 5-caffeoylquinic acid (5-CQA), together with
two major positional isomers, 4-CQA and 3-CQA (Farah &
Donangelo, 2006). The content of these isomers was determined
in the studied coffee brew and the results are displayed in Fig. 1.
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
decrease in the concentration of phenolic compounds was noted
over the period of incubation at pH 7.5 (conditions such as in the
small intestine). This study also showed a decrease in the quantity
and quality of phenolic compounds in both the coffee and cinna-
mon extracts. In turn, as Gawlik-Dziki (2004) reported, phenolic
acid contents may increase as a result of the hydrolysis of ester
bonds and glycosidic hydrolysable tannins upon heating in acidic
conditions (as is the case during digestion in vitro). In the experi-
ment the digestion process was too short-lived to cause complete
hydrolysis of the tannins present in the extract of coffee. Addition-
ally, phenolic compounds may form indigestible complexes with
proteins (Świeca, Gawlik-Dziki, Dziki, Baraniak, & Czyz,_ 2013).
their bioaccessibility. Therefore, an attempt was made to study
the LOX – inhibitory activities of this commonly-consumed bever-
age. It should be mentioned that the studied extracts of coffee and
cinnamon were a good source of potentially bioaccessible LOX
inhibitors, which were released during gastrointestinal digestion.
The results are consistent with those obtained by Gawlik-Dziki
100.00 120.00 140.00 (2012b), i.e. that in vitro pH digestion enhances the ability of the
cinnamon extract to inhibit LOX activity.
The bioaccessibility and bioavailability of dietary polyphenols
are principally relevant to their beneficial effects. Bioaccessibility
is defined as the amount of a compound released from the solid
food matrix, thereby capable of passing through the intestinal bar-
rier. However, the proportion of the compound that is digested,
absorbed, and metabolised through the normal pathways, referred
to as its bioavailability, is far more important for the nutraceutical
potential of the food (D’Archivio, Filesi, Vari, Scazzocchio, &
Masella, 2010). In order to determine the potential bioaccessibility
of compounds bearing antioxidative potential, we further analysed
the antioxidative properties of raw coffee and cinnamon extracts
(before digestion in vitro) and those extracts subjected to simu-
lated gastrointestinal processing. In turn, Saura-Calixto et al.
(2007) in their study stated that polyphenols from beverages are
completely bioaccessible, because they pass directly into the intes-
tinal fluids, and they are the largest contributors of bioaccessible
polyphenols in the small intestine. However, only a small part of
small intestine bioaccessible polyphenols can be absorbed through
the intestinal mucosa and therefore metabolised. Numerous stud-
ies have shown the bioavailability of bioaccessible polyphenols in
the small intestine to be very low, reporting values between 5%
and 10%. Therefore, in addition to polyphenols associated with
the indigestible fraction, a major part of the polyphenols bioacces-
sible in the small intestine may also reach the colon because of
their low bioavailability. Unlike in the case of synthetic pharma-
ceuticals based on the activity of single (chemical) active com-
pounds, numerous phytochemical compounds act in a beneficial
manner by an additive of synergistic activity in one or numerous
target sites connected to physiological processes. This idea has
found an application in pharmacology during investigations into
the combination of a few metabolites in multidirectional therapy
(Birskin, 2000). The method usually used for identification of inter-
actions between active compounds is isobolographic analysis. This
method is independent of activity mechanisms; however, it should
 A. Durak et al. / Food Chemistry 162 (2014) 81–88 87

5.00
4.50 A
4.00

Coffee [mg/mL]
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
0 1 2 3 4 5 6
Cinnamon [mg/mL]

2.00
B
1.50
Coffee [mg/mL]

1.00

0.50

0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
Cinnamon [mg/mL]

50.00
Chlorogenic acid [µg/mL]

40.00
C
30.00
20.00
10.00
0.00
0.00 10.00 20.00 30.00 40.00 50.00
Cinnamic acid [µg/mL]

Fig. 4. Isobole curves for 50% LOX-inhibitory activity of coffee and cinnamon mixtures – before digestion (A), after digestion in vitro (B) and mixtures of standard compounds
(C).

Table 2
Comparison of interaction factors (IF) of mixtures of coffee with cinnamon and pure chemical compounds identified in experimental material.

Sample Extracts Activity AMa ATb IF

Coffee/cinnamon mixture (1:1) Raw Antiradical potential 1.92 1.69 1.13
LOXI activity 2.72 4.60 0.59
Digested Antiradical potential 1.61 1.33 1.22
LOXI activity 1.63 1.41 1.16
Chlorogenic acid/cinnamic acid mixture (1:1) Antiradical potential 0.111 0.079 1.41
LOXI activity 0.017 0.042 0.41
a
Measured activity (expressed as EC50 [mg/mL]).
b
Theoretical calculated activity (expressed as EC50 [mg/mL]).

be emphasised that this analysis is quite complicated and labour-
consuming. Issues concerning the application of isobolographic
analysis for the determination of interactions between anti-inflam-
matory compounds (LOX inhibitors) of flowers of lime and com-
mon dandelion were undertaken in the study of Gawlik-Dziki
(2011). The first stage of the research involved a determination
of the polyphenolic profile of raw materials and a comparison of
their ability to inhibit LOX activity. Both tinctures exhibited the
ability to inhibit LOX activity in a dose-dependent manner, which
enables determination of EC50 values. The isobole adopted a con-
vex shape, which points to the fact that LOX inhibitors contained
in the examined extracts act antagonistically. In this study it was
observed that the isobole curves for the 50% LOX-inhibitory activ-
ity of coffee and cinnamon mixtures before digestion and for mix-
tures of pure chemical compounds have a concave shape (Fig. 4).
This means that both pure model compounds and the compounds
present in the tested extracts demonstrate a synergistic effect. It
should be noted, however, that the shape of the isobole curves
for coffee and cinnamon mixtures after digestion in vitro have dif-
ferent shapes, which testifies to the fact that the changes in the
extracts as a result of the digestion process cause the active com-
pounds to exert an antagonistic effect.
A definitively less complicated method for the evaluation of the
interactions between bioactive compounds was the determination
of the interaction factor (IF) proposed by Gawlik-Dziki (2012a). The
proposed way of studying interactions, such as isobolographic
analysis, is independent of mechanisms of active compound activ-
ity and requires a linear relationship between an activity and a
sample concentration. A crucial advantage is the possibility of
studying interactions between any number of components and
the fact that it is definitely less complicated. Moreover, the
‘‘strength’’ of interaction may be estimated approximately based
on the IF value. In our study, we have shown that the type of inter-
action determined on the basis of isobolographic analysis (Fig. 3
88 A. Durak et al. / Food Chemistry 162 (2014) 81–88
and Fig. 4) is confirmed by IF (Table 2). Therefore, as mentioned
above, this index can be used in the initial assessment of the inter-
action between two active ingredients.
Moreover, most studies on polyphenol bioavailability mainly
use pure single compounds (isolated from food or chemically syn-
thesised), although their bioavailability from whole foods might be
substantially different (Saura-Calixto et al., 2007). Therefore, we
wanted to compare interaction types between extracts of coffee
and cinnamon with respect to pure chemical compounds that we
used as model compounds and identified in extracts. In examining
the ABTS radical scavenging activity, isobole curves for both
extracts of coffee and cinnamon and mixtures of pure chemical
compounds were convex, which suggests that the same kind of
interaction was observed for extracts and for pure single com-
pounds. In turn, isobole curves for the 50% LOX-inhibitory activity
of raw extracts of coffee and cinnamon and for mixtures of single
chemical compounds adopt a concave shape and the kind of inter-
action is the same – synergism. Moreover, the isobole curve for cof-
fee and cinnamon after the process of digestion in vitro shows a
different kind of interaction – antagonism. This demonstrates that
interaction with the food matrix and/or changes during simulated
gastrointestinal digestion caused significant differences in rela-
tionships between the main phenolic compounds present in coffee
and cinnamon extracts.
The results suggest that the gastrointestinal tract might act as
an extractor whereby antioxidative compounds are progressively
released from the food matrix and made bioaccessible. The wide
distribution of coffee drinking impacts on a broader demographic
population than other functional foods that act on a more
defined population (Dórea & da Costa, 2005). New beneficial prop-
erties of the coffee beverage are being continuously discovered.
Furthermore, aromatic additives, such as cinnamon, are able to
change the antioxidant properties of coffee extract and antioxidant
interactions may be identified using methods of varying degrees of
difficulty. Pure chemical compounds identified in experimental
material exert different beneficial effects than coffee extract fortif-
icated with cinnamon, which means that the food matrix plays an
important role in creating the biological properties of the tested
samples.
References
Axelroad, B., Cheesborough, T. M., & Laakso, S. (1981). Lipoxygenases in soybeans.
Methods in Enzymology, 71, 441–451.
Birskin, D. P. (2000). Medicinal plants and phytomedicines. Linking plant
biochemistry and physiology to human health. Plant Physiology, 124, 507–514.
Cammerer, B., & Kroh, L. W. (2006). Antioxidant activity of coffee brews. European
Food Research and Technology, 223, 469–474.
Cheng, D. M., Kuhn, P., Poulev, A., Rojo, L. E., Lila, M. A., & Raskin, I. (2012). In vivo
and in vitro antidiabetic effects of aqueous cinnamon extract and cinnamon
polyphenol-enhanced food matrix. Food Chemistry, 135, 2994–3002.
D’Archivio, M., Filesi, C., Vari, R., Scazzocchio, B., & Masella, R. (2010). Bioavailability
of the polyphenols: Status and controversies. International Journal of Molecular
Sciences, 11, 1321–1342.
Debry, G. (1994). Coffee and health. 2-7420-0037-2. Paris: John Libbey Eurotext.
Deepa, G., Ayesha, S., Nishtha, K., & Thankamani, M. (2013). Comparative evaluation
of various total antioxidant capacity assays applied to phytochemical
compounds of Indian culinary spices. International Food Research Journal, 20,
1711–1716.
Dórea, J. G., & da Costa, T. H. M. (2005). Is coffee a functional food? British Journal of
Nutrition, 93, 773–782.
Farah, A., & Donangelo, C. M. (2006). Phenolic compounds in coffee. Brazilian Journal
of Plant Physiology, 18, 23–26.
Gawlik- Dziki, U. (2004). Phenolic acids as bioactive food ingredients. Food, Science,
Technology, Quality, 4, 29–40.
Gawlik-Dziki, U. (2012a). Changes in the antioxidant activities of vegetables as a
consequence of interactions between active compounds. Journal of Functional
Foods, 4, 872–882.
Gawlik-Dziki, U. (2012b). Dietary spices as a natural effectors of lipoxygenase,
xanthine oxidase, peroxidase and antioxidant agents. LWT – Food Science and
Technology, 47, 138–146.
Gawlik-Dziki, U. (2011). Application of izobolographic analysis for the evaluation of
interactions between bioactive constituents from dandelion (Taraxaci flos) and
lime (Tiliae flos). Annales Universitatis Mariae Curie-Skłodowska Lublin-Polonia,
XXIV, 153–160. N3, 17.
Gawlik-Dziki, U., Świeca, M., Sułkowski, M., Dziki, D., Baraniak, B., & Czyz, _ J. (2013).
Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts – In
vitro study. Food and Chemical Toxicology, 57, 154–160.
Gomez- Ruiz, J., Leake, D. S., & Ames, J. M. (2007). In vitro antioxidant activity of
coffee compounds and their metabolites. Journal of Agricultural and Food
Chemistry, 55, 6962–6969.
Hinneburg, I., Dorman, H. J. D., & Hiltunen, R. (2006). Antioxidant activities of
extracts from selected culinary herbs and spices. Food Chemistry, 97, 122–129.
Juntachote, T., & Berghofer, E. (2005). Antioxidative properties and stability of
ethanolic extracts of Holy basil and Galangal. Food Chemistry, 92, 193–202.
Mathew, S., & Abraham, T. E. (2006). Studies on the antioxidant activities of
cinnamon (Cinnamomum verum) bark extracts, through various in vitro models.
Food Chemistry, 9, 520–528.
Moselhy, S. S., & Junbi, H. H. (2010). Antioxidant properties of ethanolic and
aqueous Cinnamon extracts against liver injury in rats. International Journal of
Advances in Pharmaceutical Sciences, 1, 151–155.
Murcia, M. A., Egea, I., Romojaro, F., Parras, P., Jimenez, A. M., & Martinez-Tome, M.
(2004). Antioxidant evaluation in dessert spices compared with common food
additives. Influence of irradiation procedure. Journal of Agricultural and Food
Chemistry, 52, 1872–1881.
Oomen, A. G., Hack, A., Minekus, M., Zeijdner, E., Cornelis, C., Schoeters, G., et al.
(2002). Comparison of five in vitro digestion models to study the bioaccessibility
of soil contaminants. Environmental Science and Technology, 36, 3326–3334.
Peschel, W., Sanchez-Rabaneda, F., Dickmann, W., Plesehen, A., Gartiza, I., Jimenez,
D., et al. (2006). An industrial approach in the search of natural antioxidants
from vegetables and fruit wastes. Food Chemistry, 97, 137–150.
Re, R., Pellegrini, A., Proteggente, A., Pannala, M., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology & Medicine, 26, 1231–1237.
Record, I. R., & Lane, J. M. (1996). Simulated intestinal digestion of green and black
teas. Food Chemistry, 73, 481–486.
Sadik, C. D., Sies, H., & Schewe, T. (2003). Inhibition of 15-lipoxygenases by
flavonoids: Structure-activity relation and mode of action. Biochemical
Pharmacology, 65, 773–781.
Saura-Calixto, F., Serrano, J., & Goñi, I. (2007). Intake and bioaccessibility of total
polyphenols in a whole diet. Food Chemistry, 101, 492–501.
Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice
extracts and characterization of their phenolic constituents. Journal of
Agricultural and Food Chemistry, 53, 7749–7759.
Świeca, M., Gawlik- Dziki, U., Dziki, D., Baraniak, B., & Czyz,_ J. (2013). The influence
of protein–flavonoid interactions on protein digestibility in vitro and the
antioxidant quality of breads enriched with onion skin. Food Chemistry, 141,
451–458.
Taylor, S. R., & Demmig-Adams, B. (2007). To sip or not to sip: The potential health
risks and benefits of coffee drinking. Nutrition and Food Science, 37, 406–418.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., & Telser, J. (2007). Free
radicals and antioxidants in normal physiological functions and human disease.
The International Journal of Biochemistry & Cell Biology, 39, 44–84.
Williamson, E. M. (2001). Synergy and other interactions in phytomedicines.
Phytomedicine, 8, 401–409.
Zheng, W., & Wang, S. (2001). Antioxidant activity and phenolic composition in
selected herbs. Journal of Agricultural and Food Chemistry, 49, 5165–5170.