Determination of Caffeine Content and Antioxidant Activity of Coffee

American Journal of Applied Chemistry
2015; 3(2): 69-76
Published online March 30, 2015 (
doi: 10.11648/j.ajac.20150302.16
ISSN: 2330-8753 (Print); ISSN: 2330-8745 (Online)
Determination of Caffeine Content and Antioxidant Activity
of Coffee
Belete Tewabe Gebeyehu1, Solomon Libsu Bikila2
Department of Chemistry, Natural and computational Science College, Debre Birhan University, Debre Birhan, Ethiopia
Department of Chemistry, Science College, Bahir Dar University, Bahir Dar, Ethiopia
Email address: (B. T. Gebeyehu), (S. L. Bikila)
To cite this article:
Belete Tewabe Gebeyehu, Solomon Libsu Bikila. Determination of Caffeine Content and Antioxidant Activity of Coffee. American Journal
of Applied Chemistry. Vol. 3, No. 2, 2015, pp. 69-76. doi: 10.11648/j.ajac.20150302.16
Abstract: Attempt has been made to look into caffeine contents and antioxidant activity of coffee grown at Wembera,
Goncha, Zegie, and Burie localities of North-West Ethiopia. The caffeine content of the extracts in % w/w has been found to be
1.53 ± 0.003 for Wembera coffee, 1.41 ± 0.040 for Goncha coffee, 1.29 ± 0.033 for Zegie coffee and 0.97 ± 0.049 for Burie
coffee. The antioxidant activities of the coffee extracts were measured by using ferric reducing power assay and Rancimat
assay. Ferric reducing power assay was used to measure the total antioxidant power of water soluble components of coffee and
is expressed as ascorbic acid equivalent antioxidant capacity in milligram per gram of the dried coffee samples. The ferric
reducing power values of the extracts were 9.532 ± 0.201, 9.159 ± 0.441, 8.955 ± 0.180, 6.751 ± 0.284 for Wembera, Burie,
Goncha and Zegie coffees, respectively. The Rancimat assay was also used to measure antioxidant activity of lipid soluble
portions of coffee extracts using sunflower oil as the oxidizable substrate. It was found that all the coffee extracts improved the
oxidative stability of sunflower oil. The protection factors were 1.36 ± 0.027, 1.31 ± 0.027, 1.26 ± 0.069 and 1.17 ± 0.015 for
Wembera, Burie, Goncha and Zegie coffees respectively. Based on these results, it is suggested that Wembera coffee has the
higher caffeine content and antioxidant activities than Burie, Goncha and Zegie coffee varieties.
Keywords: Coffee, Caffeine, Antioxidant Activity, Ferric Reducing Power, Rancimat Assay
1. Introduction
The name coffee is derived from the name of the province
Keffa where shepherds from Abyssinia/Ethiopia discovered
the coffee beans in the 6th century [1]. Since then, coffee has
become one of the most widely consumed beverages
throughout the world [2] due to its pleasant taste [3], aroma
[3], [4] stimulant effect [5] and health benefits [6].
Coffee beans are the seeds of a shrub belonging to the
botanic family Rubiaceae and the genus Coffea [7]. The
generic name covers over sixty different species only three of
which, Coffea Arabica, Coffea Robusta, and Coffea Liberica
have commercial values [6]. The two most important
commercial species are Coffea Arabica and Coffea
Canephora, usually known as Arabica and Robusta varieties,
respectively [6], [8]. Arabica is considered to be a higher
quality bean, prized for its complex aroma and flavors [9],
[10] and is usually the most expensive one in the world
market [8]. Most of the coffee in the world market is
produced by developing countries including Ethiopia [9],
[11]. While most of the coffee plants cultivated in Ethiopia
are Coffee Arabica, there are, however, wide ranges of
variability among coffee cultivars in the country [1], [12],
[13]. This variability in coffee beans has been attributed to
variation in the soil, altitude and climate of the coffee
growing areas [9], [12]-[16]. These factors are believed to
influence coffees characteristics (chemical content, flavor or
aroma) [14]-[16]. For example, Brazilian researchers after
screening 300 Ethiopian coffee trees discovered three
naturally decaffeinated varieties, which they named AC1,
AC2 and AC3 [17]. Analysis of these varieties showed they
contain less than 0.07% caffeine compared to the caffeine
found in natural coffee beans [20].
Caffeine has been the subject of extensive research for its
occurrence in nature and its long history of use [21]. It is a
naturally occurring alkaloid [22] which is found in the leaves,
seeds or fruits of over 63 plants species worldwide [1], [21].
The most common sources of caffeine are coffee [12], [16],
[21], [23], cocoa beans, cola nuts and tea leaves and the
worldwide consumption of products derived from these
Belete Tewabe Gebeyehu and Solomon Libsu Bikila: Determination of Caffeine Content and Antioxidant Activity of Coffee
natural materials means that caffeine is one of the most
popular and commonly consumed drugs in the world [21].
Caffeine’s popularity stems mainly from the fact that it is a
pharmacologically active substance [24]-[26] and a mild
central nervous system stimulant [20]. Coffee has been
consumed for a long time mainly for its stimulant effects on
the central nervous system due to presence caffeine [1], [24],
[25]. However, recent literature reports associate coffee
consumption with improvement of health among humans
[23], [27]. This has been attributed to the presence of
bioactive compounds [28]-[31] in coffee which possess
antioxidant behavior [32]-[33].
Antioxidants [34] are substances that when present at low
concentrations, compared with those of the oxidizable
substrate significantly delay or inhibit oxidation of that
substrate [30], [32], [43]. In living system, antioxidants have
played an important role in scavenging excessive free
radicals [35] such as reactive oxygen species (ROS) and
reactive nitrogen species (RNS) [36]-[39] which are formed
during normal cellular metabolism in high concentration [33],
[40]. Researchers have shown that the intake of cereals, fruits,
vegetables, tea, and coffee [41], [42] are important to lower
risk of diseases that are formed as consequence of free
radicals [43]. Among those dietary species, coffee is a major
source of antioxidants and is estimated to provide half of
total antioxidant intake in several populations [20], [41], [42].
Thus, coffee extracts have received a lot of attention in recent
years and numerous studies have proven that the biologically
active components of the extracts such as chlorogenic acids
(caffeoylquinic acids [29], dicaffeoylquinic acids [29],
feruloylquinic acids
[29], [30], [31]), [34] and pcoumaroylqunic acids [29]), melanoidns [37], [39], [44] and
lipid soluble heterocyclic compounds (alkaloids) [45], [46]
play significant role for its antioxidant behavior in both vitro
and vivo [32].
The amount of chlorogenic acids, melanoids and
heterocyclic compounds (caffeine)
in coffee may be
influenced by its species, origin, brewing procedures and
roasting conditions which in turn affect the quality of its
beverages [1], [12]-[16], [23]. It is thus justifiable to attempt
to determine the caffeine content and antioxidant activity of
coffee. Among the various methods that have been employed
for the determination of caffeine in coffee, Uv-visible
spectroscopy technique is fast, simple, cheap, and available
in most laboratories [13].
Antioxidant activity of coffee can be evaluated by ferricreducing antioxidant power (FRAP) assay [47], [48]. This is
a simple, speedy and inexpensive, assay based on the ability
of phenolics to reduce Fe3+ to Fe2+ [49], [50]. The FRAP
assay is valid to quantify samples with hydrophilic
antioxidants [23]. The antioxidant activity of coffee can also
be studied by the Rancimat assay which is an automatic
determination of the oxidative stability of oils and fats
without the need for expensive and environmentaly
hazardous chemicals [49], [51], [52]. The relative activity of
the antioxidants is expressed by the protection factor,
oxidative stability or antioxidant index [53].
Geographic location is known to affect the caffeine level
and antioxidant activity [1], [12], [18], [54] of coffee
belonging to the same species [1], [12]. To the best of our
knowledge, very little research has been done on the
chemistry of coffee varieties in Ethiopia. Therefore, the aim
of the present study is to evaluate the caffeine level and the
antioxidant activity of indigenous coffee varieties produced
in four different localities (Wembera, Burie, Zegie and
Goncha) of North-West Ethiopia.
2. Materials and Methods
A. Instruments and apparatus
Traditional coffee roasting machine and electronic
blending device model-NM 8300 (Nima, Japan) were used
for roasting, grinding and homogenizing of coffee samples.
Electronic balance (Ohaus, Switzerland) was utilized to
measure mass of standards, reagents and samples. SP 65
UV/Visible spectrophotometer (SANYO, UK) was used to
measure absorbance of the samples. Filter paper (Whatman
number 542, 90 mm Dia, England), quartz cuvette, centrifuge
(West Sussex, UK), Incubator (Tuttlingen, Germany), pHmeter model 3310 (Hanna, Italy) and Rancimat Metrohm
model 743 (Herisan, Switzerland) were also used for
different purpose during the experiment.
B. Chemicals, reagents and samples
hexacyanoferrate, ferric chloride, ascorbic acid, sodium
phosphate dibasic, and sodium phosphate monobasic
dihydrate all obtained from Blulux India, caffeine standard
(St.Louis, Germany), and sunflower (Bahir Dar, local oil
factory) were used in determination of caffeine content and
antioxidant activity of coffee samples. Distilled water was
used throughout the experiment for sample preparation and
rinsing of materials. Four different coffee samples which
were grown in Wembera, Zegie, Burie, and Goncha localities
of North-West Ethiopia were collected from their production
C. Experimental procedure for determination of caffeine
by Uv-visible spectrophotometer method
Exactly 20 g of coffee from each sample was roasted by
using conventional coffee roasting machine. Each of the
roasted coffee samples was ground and screened through
250µm sieve to get a uniform texture. An accurately weighed
amount of sieved coffee (50 mg) was dissolved in 100 mL of
distilled water in a temperature range of 80-90 oC. The
solution was stirred for 30 min using magnetic stirrer and
heated gently to remove caffeine easily from the solution. In
addition the solution was filtered by using filter paper to get
rid of particle from the solution. Series of working standard
solutions of caffeine in the range of 0.016 mM to 0.102 mM
concentration were prepared in distilled water.
The extraction of caffeine from coffee has been done
according to a procedure reported by Belay [13]. In a typical
experiment, the coffee sample solution was mixed with
dichloromethane by volume ratio (25:25 mL) and the
resulting mixture was stirred for 10 min using magnetic
American Journal of Applied Chemistry 2015; 3(2): 69-76
stirrer. From the mixture, the organic phase containing most
of the caffeine (solubility of caffeine in dichloromethane is
140 mg/mL) [55] is separated from the aqueous phase with a
separating funnel. Dichloromethane is the most efficient (9899%) solvent for extraction of caffeine from coffee [56].The
aqueous phase is extracted four times with 25 mL
dichloromethane and the fractions from the organic phase
were mixed together. The absorbance of this solution was
measured in the range of 243–320 nm against the
corresponding reagent blank (dichloromethane). The
measurement of the absorbance was repeated three times for
each sample.
D. Experimental procedure for determination of antioxidant activity by FRAP assay
The reducing power was determined by the method of
Oyaizu [57] with little modification. 1 mL of different
concentration of each coffee sample extracts (1.25, 2.5, 5.0
and 10% v/v) was mixed with 2.5 mL sodium phosphate
buffer (0.2 M, pH 6.6) followed by addition of 2.5 mL of 1%
potassium hexacyanoferrate to the mixture. The mixture was
incubated at 50°C for 20 minutes then 2.5 mL of 10%
trichloroacetic acid was added to the mixture to stop the
reaction; the mixture was centrifuged 3000 rpm for 10 min.
The supernatant (2.5 mL) was mixed with 2.5 mL of distilled
water and 0.5 mL of 0.1% ferric chloride solution and
allowed to stand for 10 min. The absorbance was measured at
700 nm against the blank (distilled water) to determine the
amount of ferric ferrocyanide (Prussian blue) formed.
Series of ascorbic acid solutions in the range of 0.025
mg/g to 0.2 mg/g was used as standard to determine the
reducing power of coffee extracts. The antioxidant results
were expressed as mg of ascorbic acid equivalent antioxidant
capacity per g of coffee (dry matter).
E. Experimental procedure for determination of
antioxidant activity of coffee by Rancimat assay
5 g of each type of sieved coffee samples (Wembera, Zegie,
Burie, and Goncha) was macerated in 25 g of sunflower for
10 hours at room temperature before analysis. Samples were
centrifuged for 45 minutes at 5000rpm and the filtrate was
made ready for analysis. 3 g of each filtrate samples was
placed in four different reaction vessels and then placed in
the heating block of the wet section. In parallel to this 3 g of
sunflower oil was run as a reference in one reaction vessel.
60 mL of distilled water was also measured in five reaction
vessels containing the electrodes and placed in the heating
block of the wet section. The reaction vessels were connected
with plastic tubes with the instrument as described by
Rancimat 743 operating procedures. A temperature at 120 oC
and air flow of 20 L/h was adjusted on the computer with
Rancimat software.
F. Statistical data analysis
For the determination of caffeine content and antioxidant
activity of coffees collected from four different geographical
locations of north –west Ethiopia, all measurements and
analyses were carried out in triplicates. The results were
expressed as means ± standard error of three parallel
replicates. Analysis of variance was performed by using oneway ANOVA. The results with P < 0.05 were regarded to be
statistically significant. Data were statistically analyzed using
origin 7.0 programs.
3. Results and Discussion
A. Determination of caffeine content by Uv-visible
spectrophotmer method
The absorbance of four working standard solutions of pure
caffeine in the range of 0.016 mM and 0.102 mM was
measured at 273 nm using UV-Vis spectrophotometer. And
then the absorbance versus concentration graph (Fig.1) was
constructed to validate the UV-Vis absorption of caffeine in
terms of linearity, sensitivity, precision and for calibration
purpose to determine the caffeine content of various coffee
samples. From the calibration curve (Fig.1), the calibration
equation was: y = 10.63305x + 0.01486, R = 0.9996, SD =
0.0133, where y is absorbance, x is concentration of caffeine
and R is the linear regression coefficient. This equation
indicated that the current studies were carried out according
to the Beer’s law ranges in terms of linearity, sensitivity and
precision of the method. Thus, the proposed method allowed
for determination of caffeine in coffee samples.
Fig. 1. Calibration curve
spectrophotometer analysis
A UV-Vis spectrophotometer method cannot be used
directly for determination of caffeine in coffee beans owing
to the matrix effect of UV-Vis spectrophotometer absorbing
substances in the sample matrix [13]. In order to overcome
this difficulty the coffee samples were first dissolved in water
and then the caffeine was extracted from the solution using
dichloromethane. After extraction, the absorbance of the
solution was measured using Uv-Vis spectrophotometer
(Fig.2). As it can be seen from Fig.2, the maximum
absorbance of caffeine extracted was obtained at 276 nm.
The result (Fig.2) showed the highest amount of caffeine was
detected at Wembera coffee sample followed by Goncha,
Zegie and Burie coffees respectively.
Belete Tewabe Gebeyehu and Solomon Libsu Bikila: Determination of Caffeine Content and Antioxidant Activity of Coffee
Fig. 2. Absorbance versus wavelength of caffeine from coffee samples
The caffeine levels of the samples were calculated from
the regression equation of the best line fit (Fig. 1) of the
standards (y = 10.63305x + 0.01486, y is absorbance of
caffeine in coffee at 276 nm and x is concentration of
caffeine calculated). By using these concentrations the mass
of caffeine in coffee was determined from the relation m =
MCV (m is mass of caffeine in mg, M is molar mass of
caffeine in g/mol, C is concentration of caffeine in mM
calculated at 276 nm, V is volume of solution in mL
containing caffeine) [58]. Moreover, the percentage (w/w) of
caffeine was calculated by taking the mass of caffeine
obtained from Beer’s law and the mass of the coffee sample
on dried weight (50 mg), Table 1.
Table 1. Mass and percentage (% w/w) of caffeine in coffee sample
Coffee samples
Mass of caffeine (mg)
Caffeine (% m/m)
0.761 ± 0.004
1.53 ± 0.003
0.709 ± 0.020
1.41 ± 0.040
0.646 ± 0.016
1.29 ± 0.033
0.487 ± 0.024
0.97 ± 0.049
At 0.05 levels, the means of all the coffees are significantly different (P <
As Table 1 shows the caffeine contents(w/w) are 1.53 ±
0.003%, 1.41 ± 0.04%, 1.29 ± 0.033%, and 0.97 ± 0.049%, in
terms of mass of caffeine to mass of coffee sample for
Wembera, Goncha, Zegie and Burie coffees respectively.
There was a significant difference (P < 0.05) in caffeine
contents among all the coffee samples. This indicated that the
caffeine content of coffee beans growing in different
geographical locations was different.
In support to this study different value of caffeine contents
in coffee beans have been reported by the previous
researchers [13], [9], [59] - [61]. For example, an average
value of 1.10% by HPLC methods for 42 Ethiopian coffee
samples [9], and in the range 0.96 ± 0.01% - 1.23 ± 0.06%
for Arabic green coffee beans [59] were reported for their
caffeine contents. There are more reports that describe the
average value of caffeine to be less than 1.5% for Arabic
coffees [60]. Using derivative spectrophotometer it was also
reported that the percentage of caffeine in coffee beans was
1.36 ± 0.03% [61]. Moreover, the caffeine content in
Ethiopian Arabica coffee grown in Bench Maji, Gediyo
Yirgachefe, Tepi, and Godere has been determined by UV-Vis
spectroscopy to be 1.1 ± 0.01%, 1.01 ± 0.04%, 1.07 ± 0.02%,
and 1.19 ± 0.02%, respectively [13]. Therefore, these values
are in reasonable degree of agreement with the findings of
the present work.
In summary, the caffeine level of coffee beans produced in
the study areas decreased in the order of Wembera, Goncha,
Zegie to Burie (Table 1). All the coffee samples have the
caffeine level in the range of the caffeine contents of different
coffee Arabica reported previously [9], [13], [60]-[61]. Even
though all the samples were roasted between 8-12 minutes
until the medium level roasting; the literatures [59] also
describe roasting does not significantly affect the content of
caffeine other than causing a slight relative increase due to
the loss of other component. Hence the variation in caffeine
level of coffee samples may be due to geographical origins
which might have different altitude, soil type, rain fall and
other agricultural as well as environmental conditions. The
variations in caffeine content of coffees are well documented
in literatures [20], [23].
A. Ferric reducing antioxidant power assay
A calibration curve was obtained using four different
concentrations of ascorbic acid in the range of 0.025- 0.2
mg/mL for the determination of the antioxidant reducing
power of coffee extracts. The equation of the calibration
curve was obtained from the resulting absorbance versus
concentration curve and the equation used was: y = 3.528x +
0.087; R = 0.978. Where: y is the absorbance at 700nm, x is
concentration of ascorbic acid in mg/mL and R is linear
regression coefficient.
As the confirmatory test for the reducing ability of coffee
extracts an aqueous solution of ferric chloride was mixed
with the reaction mixture after incubation at 50 oC for 20 min.
It was observed that the color of the solution changed from
yellowish to Prussian blue. This is an indication for the
reduction of Fe+3 to Fe+2 that might be occurred due to the
presence of water soluble antioxidants components of coffee
[20]. The formation of Fe+2 can be monitored by measuring
the absorbance of Prussian blue at 700 nm [49].
As it was observed during the experiments, there was a
variation in the intensity of the color among the different
concentrations of coffee extracts. The color of the complex
was varied in the range of lighter Prussian blue to deep blue
when the concentrations of coffee extracts were changed
from 1.25%, 2.5%, 5% to 10% (v/v) and also there was a
difference among the coffee extract types of the same
concentration. As shown in Fig.2, the absorbance of the
Prussain blue complex was changed with the difference in
concentrations and coffee varieties. In all of the coffee
extracts, the values of the absorbance were increased with the
increase in concentration of the samples (Fig.3). Moreover,
the absorbance of the extracts decreased in the order of
Wembera, Burie, Goncha to Zegie coffees (Fig.3). Since an
American Journal of Applied Chemistry 2015; 3(2): 69-76
increase in absorbance is indicative for the higher reducing
power of coffee extracts, Wembera coffee would have the
highest ability in reduction of ferric to ferrous form while
Zegie coffee extract has the lowest one (Table 2).
Fig. 3. Absorbance versus concentration of samples
The ferric reducing antioxidant power of coffee extracts
was expressed in terms of ascorbic acid equivalent
antioxidant capacity [62]. Briefly, the results were expressed
as the maximum concentration of coffee extract having ferric
reducing ability equivalent to that of 0.2 mg/mL of ascorbic
acid, particularly expressed as milligram of ascorbic acid
equivalent per gram of coffee sample in the dried weight. The
antioxidant activity of coffee extracts was also described as
the relative percentage reducing power of extracts in
comparison with ascorbic acid [63] the results are
summarized in Table 2.
Table 2. FRAP values and percentage reducing power (%RP) of coffee
of coffee
FRAP values
in AEAC (mg/g
Reducing power
(% RP)
9.532 ± 0.201 a
77.907 ± 1.57 a
9.159 ± 0.441
75.253 ± 0.51 a
8.955 ± 0.180 a
73.947 ± 3.42 a
58.463 ± 0.52 b
6.751 ± 0.284
Values are mean ± standard deviation of triplicate measurements (n = 3).
Values of the column followed by the same letter indicates no significant
difference (p >0.05) and different letters are significantly different (P < 0.05).
Table 2 shows that the reducing power of coffee extracts
varied with the variation in the coffee samples. Particularly,
Wembera coffee exhibited relatively strong reducing power
(better able to donate an electron) in comparison with other
extracts, which may be due to its high contents of water
soluble antioxidants. Literatures reveal coffee beans contain
efficient water soluble antioxidants, such as chlorogenic
acids, caffeic acids, ferulic acids, p-coumaroic acids,
melanoids and alkaloids;[20], [38], [39], [46] and their
content depends mainly on the coffee species, origin and
degree of roasting [13], [23], [44], [54], [64]. Hence the
coffee samples in this study were roasted in same way, the
variation in their reducing power might be due the difference
in their geographical locations (origin) and their species. In
agreement with the literatures; the results of the study (Table
2 and Fig.3) also revealed that coffee samples from various
geographical locations of North- West Ethiopia have
possessed different antioxidant activities.
B. Determination of antioxidant activity by Rancimat assay
The oxidative stability of sunflower oil in the presence and
absence of different coffee samples was determined by
measuring its induction time using Rancimat assay [52].
When coffee extracts were exposed to a stream of airflow 20
L/h at temperature 120 °C, the volatile oxidation products
were transferred to a measuring vessel by the air stream and
absorbed in the measuring solution (distilled water). As the
conductivity of this measuring solution was recorded
continuously its induction time was recorded automatically
and the mean values are shown in Table 3.
It was observed that in rapid production of volatile acids at
the end of the induction time, expressed in hours, induces an
increase of the water conductivity. The induction time of the
substrates with coffee extracts were: 2.670 ± 0.010, 2.573 ±
0.100, 2.480 ± 0.185 and 2.290 ± 0.080 h for Wembera, Burie,
Goncha and Zegie coffees respectively (Table 3).
As shown in Table 3, the time required for the formation of
a sufficient concentration of initiating radicals was greater
when the coffee samples were added to the substrate,
delaying the onset of the propagation phase of the radical
chain reaction [65]. This might be happened due to the
presence of different concentration of lipophilic antioxidants
such as polyphenolic acids, melanoids and heterocyclic
compounds [38], [39] in coffee extracts that slow down the
oxidation process of the substrate through donation of
hydrogen atom.
According to statistical analysis of variance (Table 2),
there was a significant difference (P < 0.05) between Zegi
with Wembera and Burie coffees to delay the oxidation of
substrate. This shows that Zegie coffee might have different
antioxidant activity from Wembera and Burie coffees. The
values of protection factors were increased from Zegie,
Goncha, Burie to Wembera coffees as shown in Table3.
Hence, PF >1and its values are different, coffee samples
added might delay the formation of hydroperoxides within a
different rate by offering various protection for the substrate
[65]. Particularly, Wembera coffee was better in providing
protection for the substrate from being oxidized. This might
be due to the difference in concentrations of lipid soluble
antioxidant components of coffee.
Similar to the results of present findings, researcher using
Rancimat assay indicates that coffee beverages from different
geographical locations have offered different protection to
the oxidation stability of the substrates (oil and butter) [64].
The variations in protection factors have been explained due
to the difference in concentrations of lipid soluble
constituents of coffee extracts like polyphenolic acids,
Belete Tewabe Gebeyehu and Solomon Libsu Bikila: Determination of Caffeine Content and Antioxidant Activity of Coffee
melanoids and volatile heterocyclic compounds [39].
Table 3. Induction time (IT (h)) and protection factor (PF) values of coffees
with sunflower oil.
Samples and sunflower oil
IT (h)
Wembera coffee
2.670 ± 0.010
1.36 ± 0.027
Burie coffee
2.573 ± 0.100
1.31 ± 0.027
Goncha coffee
2.480 ± 0.185
1.26 ± 0.069
Zegie coffee
2.290 ± 0.080
1.17 ± 0.015
Sunflower oil
1.963 ± 0.041
Values are mean ± standard deviation of triplicate measure ments (n = 3).
Inspite of the experimental differences, the Rancimat assay
and the FRAP assay were found to show similar trends when
comparing extracts of different coffee samples in their
ranking order of antioxidant activities. In both cases the
antioxidant activities of coffee samples were in the
decreasing order of: Wembera Burie, Goncha to Zegie
coffees. But there was a slight difference in their significant
level. This might be the difference in concentration of water
and lipid soluble antioxidant components of coffees.
On the other hand, as it was revealed in this study the
caffeine level of coffee samples were in a decreasing order of:
Wembera, Goncha, Zegie to Burie coffees. This indicates that
Burie coffee extract possesses the least value of caffeine
content but the second higher value in its antioxidant activity.
And also Zegie coffee has higher caffeine content than Burie
coffee but the lower in its antioxidant activity. This shows
that caffeine might not be the major antioxidant components
of coffee and its role is almost negligible and does not have
significant contribution to the overall antioxidant activity of
In light of this study results all coffee samples have higher
antioxidant activities in both assays. The high antioxidant
efficiency of coffee might be attributed to the high content of
water soluble and lipid soluble compounds such as
chloronogic acids, caffeic acids, melanoids and heterocyclic
compounds present in coffee. The presences of these
compounds and their antioxidant activity have already been
reported by the previous researchers [20], [32], [31], [36],
samples of coffee in both assays were comparable. The ferric
reducing power values of water soluble portions of coffee
extracts in terms of ascorbic acid equivalent antioxidant
capacity were 9.532 ± 0.201, 9.159 ± 0.441, 8.955 ± 0.180,
6.751 ± 0.284 mg/g dried weight for Wembera, Burie,
Goncha and Zegie coffee, respectively. In a similar note, lipid
soluble portions of coffee extracts using Rancimat assay
improved the oxidative stability of the substrate (sunflower
oil) from being oxidized by delaying the onset of propagation
through donation of hydrogen. The protection factors
(oxidative stability indexes) were 1.36 ± 0.027, 1.31 ± 0.027,
1.26 ± 0.069 and 1.17 ± 0.015 for Wembera, Burie, Goncha
and Zegie coffees respectively.
In both cases Wembera coffee has shown the highest value
of caffeine content and antioxidant activity among the other
coffee varieties. However, the trend in caffeine content and
antioxidant activities of Burie, Goncha and Zegie coffee
extracts are not proportional. This study shows that the
consumption of coffee with higher caffeine content might not
be directly related with the benefits of higher antioxidant
activities. Thus, the antioxidant behavior of coffee is mainly
attributed by other compounds like chlorogenic acids, ferulic
acids, caffeic acids and melanoids.
In light of the results of the present study, it may be
suggested that normal dose coffee intake may exert several
health beneficial effects by virtue of its antioxidant
constituents which protect the body against diseases caused
by oxidative stress. These findings warrant the need to
further carry out similar studies so as to promote cultivation
of those coffee plants most promising for health advancement.
As caffeine contents and antioxidant activities of the coffee
samples varied on the basis of geographical locations; there
should be further study on agricultural and environmental
factors that resulted in these differences. Since antioxidant
activities of coffee extracts were tested only with the FRAP
and Rancimat assays, there should be a need to test with
other assays.
T. Ranheim, B. Halvorsen, “Coffee consumption and human
health-beneficial or detrimental?Mechanisms for effects of
coffee consumption on different risk factors for
cardiovascularndisease and type 2 Diabetes mellitus,”
Molecular Nutrition and Food Research, 49, 274-284, 2005.
M. R. Camargo, C. F. Toledo, G. Farah. “Caffeine daily intake
from dietary sources in Brazil,” Food Additives Contaminants,
16, 79–87, 1999.
S. Schenker, C. Heinemann, M. Huber, R. Pompizzi, R. Perren
and F. Escher, “Impact of roasting conditions on the formation
of aroma compounds in coffee beans,” Journal of Food
Science, 67, 60-66, 2002.
A. M. Costa, C. Parreira, and L. Vilas-Boas, “The use of an
electronic aroma sensing device to assess coffee
differentiation comparison with SPME gas chromatography
mass spectrometry aroma patterns,” Journal of Food
Composition and Analysis, 14, 513-522, 2001.
4. Conclusion
The objective of this study was to determine the caffeine
content and antioxidant activities of coffee varieties obtained
from four different localities of North-West Ethiopia. The
caffeine contents (%w/w) of coffee samples grown in
Wembera, Goncha, Zegie, and Burie have been found to be
1.53 ± 0.003%, 1.41 ± 0.04%, 1.29 ± 0.033% and 0.97 ±
0.049%, respectively, suggesting dependence of caffeine
content on the location where the coffee plants are grown.
FRAP and Rancimat assays were employed for the
determination of antioxidant activities of the coffee samples
obtained from the four localities of North-West Ethiopia. The
results show that the trends in antioxidant activities of the
American Journal of Applied Chemistry 2015; 3(2): 69-76
A. Aresta, F. Palmisano, C. G. Zambonin, “Simultaneous
determination of caffeine, theobromine, theophylline,
paraxanthine and nicotine in human milk by liquid
chromatography with diode array UV detection,” Food
Chemistry, 93, 177–181, 2005.
[23] C. W. Huck, W. Guggenbichler, G.K. Bonn, “Analysis of
caffeine, theobromine and theophylline in coffee by near
infrared spectroscopy (NIRS) compared to HPLC coupled to
mass spectrometry,” Analytica Chimica Acta., 538, 195–203,
V. J. Higdon, B. Frei, “Coffee and health: a review of recent
human research,” Crit. ReV. Food Sci. Nut., 46, 101-123,
M. N. Clifford, C. L. Gibson, J. J. R. Rakotomalala, E. Crost,
A. Charrier, “Caffeine from green beans of Mascarocoffea,”
Phytochemistry, 30, 4039-4040, 1991.
[24] A. A. P. Almeida, A. Farah, D.A.M. Silva, E.A. Nunan, M. B.
A. Glória, “Antibacterial activity of coffee extracts and
selected coffee chemical compounds against enterobacteria,” J.
Agric. Food Chem., 54, 8738-8743, 2006.
M. J. Martin, F. Pablos, A. G. Gonzalez, “Characterization of
green coffee varieties according to their metal content,”
Analytica Chimica Acta., 358, 177-183, 1998.
D.Yigzaw, M.T. Labuschagne, G. Osthoff, L. Herselman,
“Variation for green bean caffeine, chlorogenic acids, sucrose
and trigonelline contents among Ethiopian Arabica coffee
accessions,” SINET: Ethiopian Journal of Science, 30, 77-82,
[10] J. Gray, “Caffeine, coffee and health,” Nutrition and Food
Science, 6, 314-319, 1998.
[11] K. F. Wiersum, T.W. Gole. “Certification of wild coffee in
Ethiopia: Experiences and challenges,” 2008.
[12] J. J. Barone, H. R. Roberts “Caffeine consumption,” Food
Chem. Toxicol., 34, 119–129, 1996.
[13] A. Belay, K. Ture, M. Redi, A. Asfaw, “Measurement of
caffeine in coffee beans by UV- Vis spectrometer,” Food
chem., 108, 310-315, 2008.
[14] R. P. Heaney, “Effects of caffeine on bone and the calcium
economy,” Food and Chemical Toxicology, 40, 1263-1270,
[15] D. Perrone, C. M. Donangelo, A. Farah, “Fast simultaneous
analysis of caffeine, trigonelline, nicotinic acid and sucrose in
coffee by liquid chromatography–mass spectrometry,” Food
Chem., 110, 1030–1035, 2008.
[16] H. Ashihara, H. Sano, A. Crozier, “Caffeine and related purine
alkaloids: Catabolism, function and genetic engineering,”
Phytochemistry, 69, 841-856, 2008.
[17] M. B. Silvarolla, P. Mazzafera, L.C. Fazouli, “A naturally
decaffeinated arabica coffee,” Nature, 429, 826, 2004.
[18] I. Hecimovic, A. Belscak-Cvitanovic, D. Horzic, D. Komes,
“Comparative study of polyphenols and caffeine in different
coffee varieties affected by the degree of roasting,” Food
Chem., 129, 991-1000, 2011.
[19] K. Singh, A. Sahu, “Spectrophotometer determination of
caffeine and theoylline in pure alkaloids and its application in
Pharmaceutical formulations,” Anal. Bio. Chem., 349, 176180, 2006.
[20] A. Svilaas, A. K. Sakhi, L. F. Andersen, T. Svilaas, E. C Strom,
J. D. R. Jacobs, L. Ose, R.Blomhoff, “ Intake of antioxidants
in coffee; wine and vegetables are correlated with plasma
carotenoids in human,” J. Am. Nutr. Sci., 134: 562-567, 2004.
[21] S. Kolayli, M. Ocak, M. Kucuk, R. Abbasoglu, “Does caffeine
bind to metal ions?” Food Chem., 84, 383-388, 2004.
[22] P. M. Dewick, “Medicinal natural products: a biosynthetic
approach” England: John Willey and Sons, 392-396, 2001.
[25] C. A. Brown, C. Bolton-Smith, M. Woodward, M. H. TunstallPedoe, “Coffee and tea consumption and the prevalence of
CHD in men and women: results from the Scottish Heart
Health Study,” Journal of Epidemiological Community Health,
47, 171-175, 1993.
[26] Z. Textor, M. Beer, M. Anetseder, H. Ko-Stler, E. Kagerbauer,
W. Kenn, D. Hahn, N.Roewer, “Caffeine impairs
intramuscular energy balance in Patients,” Muscle Nerve, 28,
353- 36, 2003.
[27] R. L. Prior, X. Wu, K. Schaich, “Standardized methods for the
determination of antioxidant capacity and phenolics in foods
and dietary supplements,” J. Agric. Food Chem., 53, 42904302, 2005.
[28] J. M. Tunnicliffe, J. Shearer,“Coffee, glucose homeostasis,
and insulin resistance: physiological mechanisms and
mediators,”Appl. Physiol. Nutr. Metab., 33, 1290–1300, 2008.
[29] G. S. Duarte, A.A. Pereira, A. Farah, “Chlorogenic acids and
other relevant compounds in Brazilian coffees processed by
semi-dry and wet post-harvesting methods,” Food Chem., 118,
851-855, 2010.
[30] M. N. Clifford, “Chlorogenic acid and other cinnamatesnature, occurrence, dietary burden, absorption and
metabolism,” J .Sci. Food Agric., 80, 1033-1043, 2000.
[31] V. D. Truong, R. F. Mcfeeters, R .T. Thompson, L. L. Dean, B.
Shofran, “ Phenolic acid content and composition in leaves
and roots of common commercial Sweet potato(Ipomea
batatas L.) Cultivar in the United States,” J. Food. Sci., 72,
343-349, 2007.
[32] A. Gomez-Ruiz, M. Ames, S. Leake, “Antioxidant activity
and protective effects of green and dark coffee components
against human low density lipoprotein oxidation,” Eur Food
Res. Technol., 22, 1017–1024, 2008.
[33] R. H. Stadler, R. J. Turesky, O. Mller, J. Markovic, P. M.
Leong-Morgenthaler, “The inhibitory effects of coffee on
radical-mediated oxidation and mutagenicity,” Mutat. Res.,
308, 177–190, 1994.
[34] A. Stalmach, W. Mullen, C. Nagai, A. Crozier, “On-line HPLC
analysis of the antioxidant activity of phenolic compounds in
brewed, paper-filtered coffee,” Brazilian Journal of Plant
Physiology, 18, 253–262, 2006.
[35] M. Namiki, “Antioxidants/antimutagens in food,” Crit.Rev.
Food Sci.Nutr., 29, 273-300, 1990.
[36] M. D. del Castillo, J. M. Ames, M. H. Gordon, “Effect of
roasting on the antioxidant activity of coffee brews,” J. Agric.
Food Chem., 50, 3698–3703, 2002.
[37] K.W. Kang, S. J. Oh , S. Y. Ryu, G.Y. Song, B. H. Kim, J. S.
Kang, S. K. Kim, “Evaluation of the total oxy-radical
scavenging capacity of catechins isolated from green tea,”
Food Chem., 121, 1089-1094, 2010.
Belete Tewabe Gebeyehu and Solomon Libsu Bikila: Determination of Caffeine Content and Antioxidant Activity of Coffee
[38] A. Farah, T. de Paulis, D. P. Moreira, L. C. Trugo, P. R. Martin,
“ Chlorogenic acids and lactonesin regular and water
decaffeinated arabica coffees” J. Agric. Food Chem., 54, 374–
381, 2006.
[39] S. Shizuuchi, F. Hayase, “Antioxidative activity of the blue
pigment formed in a D-xylose-glycine reaction system,”
Biosci. Biotechnol. Biochem., 67, 54–59, 2003.
[40] J. M .McCord, “The evolution of free radicals and oxidative
stress,” Am. J. Med., 108, 652-659, 2000.
[41] J. G. Dorea, T. H. M. da Costa, “Is coffee a functional food?”
British Journal of Nutrition, 93, 773-782, 2005.
[42] R. Pulido, F. Saura-Calixt, “ Contribution of beverages to the
intake of lipophilic and hydrofilic antioxidants in the Spanish
diet,” European Journal of Clinical Nutrition, 57, 1275–1282,
[43] A. Podsędek, “Natural antioxidants and antioxidant activity of
Brassica vegetables:” A review LWT-Food Sci. Technol., 40,
1-11, 2007.
[44] C. Delgado-Andrade, A. Josea, R. Henares, J. Morales,
“Assessing the Antioxidant Activity of Melanoidins from
Coffee Brews by Different Antioxidant Methods,”J. Agric.
Food Chem., 53, 7832-7836, 2005.
[45] K. Yanagimoto, H. Ochi, K. G. Lee, T. Shibamoto,
“Antioxidative activities of fractions obtained from brewed
coffee,” J. Agric. Food Chem., 52, 592–596, 2004.
[46] S. Azam, N. Hadi, N. U. Khan, S. M. Hadi, “Antioxidant and
prooxidant properties of caffeine, theobromine and xanthine,”
Med. Sci. Monit., 9, 325–330, 2003.
[47] K. Fujioka, T. Shibamoto, “Chlorogenic acid and caffeine
contents: In various commercial brewed coffees,” Food Chem.,
2008, 106, 217-221.
[48] N. Hunda-Faujan, A.Noriham, A.S. Norrakiah, and A. S.Babji.,
“Antioxidant activity of plants methanoic extracts containing
phenolic compounds,”Afr.J.Biotechnol., 8, 484-489, 2009.
[49] I. F. F. Benzie and J. J. Strain, “The ferric reducing ability of
plasma (FRAP) as a measure of antioxidant power: The FRAP
assay,” Analytical Biochemistry, 1996, 239, 70–76.
[50] K. I. Berker, K. Güçlü, I.Tor, R. Apak, “ Comparative
Evaluation of Fe(III) Reducing Power-Based Antioxidant
Capacity Assays in the Presence of Phenanthroline, Bathophenanthroline,Tripyridyltriazine(FRAP), and Ferricyanide
Reagents,” Talanta., 72, 1157-1165, 2007.
[51] M. H. Gordon, E. Mursi, “A comparison of oil stability based
on Metrohm Rancimat with storage at 20 oC,” J. AOCS., 71,
649-651, 1994.
[52] J. O. Ragnarsson, T. P. Labuza,” Accelerated shelf life test for
antioxidantive rancidity,” Food Chem., 2, 291–308, 1977.
[53] A. M. Vera, M. A. Martinez-Tome, M. Jimenez, A. Jimenez,
M. Honrubia, “Antioxidant activity of edible fungi (truffles
and mushroom): losses during industrial process” Journal of
food protection, 65, 1614-1622, 2002.
[54] R. Amarowicz,“Antioxidant activity of Maillard reaction
products,” Eur. J. Lipid Sci. Technol, 111, 109–111, 2009.
[55] B. Stavric, “Methylxanthines: toxicity to humans,” Food
Chem Toxicol., 26, 645-662, 1988.
[56] M. Haenen, H. V. den Berg, A. Bast, “Applicability of an
improved Trolox equivalent antioxidantcapacity (TEAC)
assay for evaluation of capacity measurements of mixtures,”
Food Chem., 66, 511-517, 1999.
[57] M. Oyaizu, “Studies on products of browing reactions:
Antioxidant activities of products of browing reaction
prepared from glucosamine,” Jpn. J. Nutr., 1986, 44, 307-315.
[58] D. Harvey, Modern Analytical Chemistry. Mc Graw Hill, 1st
edition, 15-18, 2000.
[59] A. Farah, M. C. M Monteiro, V. Calado, A. S. Franca, L. C.
Trugo, “Correlation between cup quality and chemical
attributes of Brazilian coffee,” J. Food Chem., 98, 373–380,
[60] E. Illy, “The complexity of coffee,”Scientific America, 86-91,
[61] G. Alpdogan, K. Karbina, S. Sungur, “Derivative
spectrophotometer determination of caffeine in some
beverages,” Turkish Journal of Chemistry, 26, 295–302, 2002.
[62] S. Coban, “Development of Biosensor for determination of the
total antioxidant capacity,” Thesis, Izmir Institute of
Technology, 132-135, 2008.
[63] A. Rohman, S. Riyanto, N. Yuniarti, W. R. Saputra, R. Utami,
and W. Mulatsih, “Antioxidant activity, total phenolic and
total flavaonoid of extracts and fractions of red fruit
(Pandanus conoideus Lam),” International Food Research
Journal, 17, 97-106, 2010.
[64] N. V. Yanishlieva, and E. M. Marinova, “Antioxidant
effectiveness of some natural antioxidantsin sunflower oil,”
Fur Lebensmittel-Untersuchung und Forschung, 203, 220-223,
[65] M. Martinez-Tome, A. M. Jimeinez, S. Ruggieri, N. Frega, R.
Strabbioli, M. A. Murcia, “Antioxidant properties of
Mediterranean spices compared with common food
additives.,” Journal of Food Protection, 64, 1412–1419, 2001.