Berhan International Research Journal of Science and Humanities

(BIRJSH), 2023, 7(1), 1–16

Journal homepage: www.journals.dbu.et ISSN: 2414-2794

Comparative Analysis of Caffeine Level in Marketed Ethiopian Tea and

Coffee Samples
Destaw Abebe1, Minbale Gashu1*

1
Debre Berhan University, College of Natural and Computational Science, Debre Berhan, Ethiopia
DOI: https://doi.org/10.61593/DBU.BIRJSH.01.07.01

Abstract

Tea (Camellia sinensis) and Coffee (Coffea arabica) are the most popular drinks next to
water for their stimulant activity. They contain different metabolites that are beneficial to
human health including the common and the psychoactive caffeine. Therefore, assessment of
the caffeine level in such natural resources is used as a tool for evaluating their quality. The
aim of this study was comparing the caffeine level of tea leaves and roasted coffee beans
grown and marketed in Ethiopia. In this work, the standard caffeine isolated from both tea
leaves and coffee beans using chromatographic techniques was characterized using
spectroscopic techniques. The caffeine contents of the extracts from tea leaves and roasted
coffee beans were analyzed using UV-Vis spectrophotometer and found 90.75 ppm and 94.25
ppm, respectively. Antibacterial activity assay of isolated caffeine against Escherichia coli,
Staphylococcus aureus, Salmonella typhi, and Listeria monocytogen was also evaluated and
the results showed its potential activity against most tested bacteria.

Keywords: Tea, Coffee, Caffeine, UV-Vis spectrophotometer, Debre Berhan

*
Corresponding author email: [email protected]
Article information: Received November 15, 2023; Revised 19 December 2023; Accepted 21 December 2023
© 2023 Debre Berhan University. All rights reserved.

Introduction bioactive compounds responsible for tea’s

Tea (Camellia sinensis) and Coffee health benefits. Caffeine is one of the

(Coffea arabica) are frontline commodity alkaloid constituents of tea besides

crops in Ethiopia (Dechassa et al, 2022; phenolic compounds (Mohammed et al,

Degaga,2020; Amamo, 2014) and the most 2009; Tiwari et al, 2011; Chen et al, 2012;

popular beverages consumed worldwide Farah, 2012; Kreicbergs et al, 2011;

for their stimulant activity. Tea is Mussatto et al, 2011).

originated from China as a medicinal drink

and it becomes popular in the rest of the Ethiopia is considered as a primary center

world including Ethiopia (Bhoo-Pathy, et of genetic diversity of coffee plants where

al, 2015) due to the phenolics as the major the name coffee was coined from the area

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 Abebe and Gashu, BIRJSH, 2023, 7(1), 1–16

locally named as Kaffa, and it was then have been recognized as important
introduced into the globe. Caffeine found attributes of their sensory quality
in tea and coffee determine the (Minbale, 2009). Most of the aroma is due
characteristic flavor and stimulant effect of to the volatile coffee oil fraction, which
tea and coffee (Mussatto et al, 2011; comprises about 10% of the roasted beans.
Adepoju et al, 2017; Minbale, 2009) The volatile oil contains mainly ethyl
together with other constituents. Arabica acetate and furfural with traces of several
coffee with its lower sourness, bitterness other compounds (Minbale, 2009). The
and better flavor is the most cultivated and fixed oil fraction of coffee oil is non-
more liked by consumers (Minbale, 2009). volatile. The most important compounds in
Robusta coffee beans contained higher coffee that is relevant for flavor and aroma
concentration of caffeine and chlorogenic are caffeine, chlorogenic acid, trigoneline,
acid compared with Arabica, while and sucrose. Carboxylic acids are
Arabica is richer in trigonelline. Beans of responsible for sourness while chlorogenic
Arabica and Robusta contain about 1% acid and caffeine determine bitterness
and 2% caffeine, respectively (Minbale, (Minbale, 2009; Mumin et al, 2006;
2009; Mumin et al, 2006; Samiullah et al, Samiullah et al, 2015; Parvathy et al, 2014;
2015). Gopinandhan et al, 2014; Grujić-Letic et
al, 2016).
The tea leaves and roasted coffee beans are
brewed, and commonly consumed as tea Excessive use of caffeine can cause
and coffee beverages, respectively. They adverse side effects, anxiety, depression,
contain different metabolites that are difficulty sleeping, nausea, restlessness,
beneficial to human health including their tremors, a fast heart rate, irritability,
popular caffeine which is responsible for increasing of blood pressure, and disrupt
their psycho-activity. Assessment of the normal heart rhythm. The consumption of
caffeine level in tea leaves and coffee caffeine containing beverages such as tea
beans grown in Ethiopia is crucial to their and coffee should be limited. In order to
quality and this was the focus of this study. aware consumers and the scientific

In the roasting process, characteristic odor community, the study focused on the
2
and flavor are developed together with the analysis of the caffeine content of some tea
and coffee samples collected from Debre
formation of dark brown color. Aroma,
sourness and bitterness of coffee brews Berhan.

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 Abebe and Gashu, BIRJSH, 2023, 7(1), 1–16

Materials and Methods using separatory funnel thrice. The

Sample collection and preparation extracts were dried by using anhydrous

sodium sulphate and dichloromethane was
Powdered, marketed and packed tea and removed by rotary evaporator to afford
coffee samples were purchased from crude caffeine. The crude caffeine was
Debre Berhan (Ethiopia), respectively in further purified using recrystallization
March 2018 and they were stored in from ethanol (97%). Finally, the purified
bottles until use. They were used as white crystals were air dried and weighed.
purchased without further modification. Pure caffeine was submitted to NMR
analysis (Addis Ababa University, AAU)
Chemicals and equipment used
and it was used further as a reference in
In this study, organic solvents like acetone,
UV-Vis quantitative analysis. The isolated
chloroform, hexane, dichloromethane,
caffeine was also analyzed by Infrared
ethyl acetate, methanol, and ethanol were
spectrometer in the range 400-4000cm-1
used besides sodium carbonate, and
using the KBr pellet technique in AAU.
anhydrous sodium sulfate as purchased.
The apparatus used in this study were
Thin layer chromatographic analysis
electric grinder, magnetic stirrer, and
electronic balance, heating mantle, The isolated caffeine from both samples in
separatory funnel, rotary evaporator and dichloromethane was analyzed using silica
UV-Vis spectrophotometer (model UV- gel thin layer plates and developed using a
1700). solvent system: ethyl acetate, methanol
Isolation of caffeine from tea and coffee and water (100:13.5:10). The thin layer
samples and its characterization plate was first sprayed with 0.5 g
Caffeine was isolated from tea and coffee potassium iodide and 0.5 g iodine
samples using the following method. The dissolved in 50 ml ethanol, followed by
mixture of tea or coffee samples (50 g) spraying with a 1:1 mixture ratio of 25%
placed into 1000 ml beaker filled with 875 HCl: 96% ethanol [11]. The caffeine zone
ml of distilled water and 10 g of sodium was indicated by a dark-brown color
carbonate were boiled for 10 min and noticed in visible light. The retention time
3
cooled on ice bath. After filtration of the (RF) of both tea and coffee extracts were
residues, the organic layer compared with that of the reference
(dichloromethane phase) was separated caffeine.

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Sample preparation for UV-Vis and Staphylococcus aureus were obtained

Analysis: Extracted caffeine (250 mg) was from Department of Biology (Debre
taken in to 250 ml flask containing 20 mL Berhan University) and antibacterial assay
of distilled H2O. Then 0.01 M HCl was accomplished using a modified agar
solutions were prepared and 10 ml of this diffusion method. Nutrient agar was
was added to the water and caffeine inoculated with microbial cell suspension
mixture in 250 ml flask. Lead acetate 2 mL (200 µl in 20 ml medium) and poured into
of 1M was added to the volume and it was sterile Petri dishes. Both compounds
made up to the mark by adding distilled extracted from black lion coffee and tea
water. The solution was shaked for 1 min was dissolved in DMSO to reach a final
and filtered. 4.5 molar solution of concentration of 100 mg/ml, 50 mg/ml, 25
sulphuric acid (H2SO4) was prepared and mg/ml and 12.5 mg/ml, to be tested. A
0.2 mL of this was added into the filtrate. standard 6 mm disc containing gentamycin
The extracted solution for both tea and (Bioanalyse) 10 μg/disc was used as the
coffee and standard dilutions were positive control. After pre-incubation for 2
analyzed at 274 nm through UV-Vis hours in a refrigerator, the plates were
spectrophotometer (Atomssa et al, 2011; incubated overnight at 37°C for 24 hours.
Belay et al, 2011). At the end of the incubation period,
antibacterial activity was evaluated by
The stock solution of standard caffeine
measuring the zones of inhibition
was prepared by adding pure caffeine (100
(Nonthakaew et al, 2015).
mg) into distilled water (100 mL), from
which working solutions of different Results and Discussion
concentrations were prepared in order to
Isolation of caffeine from tea and coffee
get calibration curve by taking 0,
20,40,60,80, 100 and 120 ppm (mg/L) in Extraction of tea (1-13B) and coffee (1-
10B) using dichloromethane as an
volumetric flask (25 mL) with the addition
extracting solvent resulted in crude
of HCl (0.1 mL) in each flask and filled
the flask with distilled water up to the caffeine content of 3.2% and 3.6%,

mark (Atomssa et al, 2011). respectively (Table 1). The amount of pure
caffeine obtained after purification using 4
Antibacterial activity of caffeine 97% of ethanol was 2.7% (tea) and 2.8%
Four bacteria, namely, Escherichia coli, (coffee).
Salmonella thyphi, Listeria monocytogen
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 Abebe and Gashu, BIRJSH, 2023, 7(1), 1–16

Table 1. Percentage of Caffeine from Tea and Coffee

Sample Sample (g) Amount of caffeine Crude caffeine Caffeine (g), caffeine %
extracted (g) in extract, % after purification (g)
1-13B 50 1.6 3.2 1.3, 2.7
1-10B 50 1.8 3.6 1.4, 2.8

Caffeine isolated from both 1-13A and1-

Characterization of caffeine
10B were observed at Rf value (0.67) and
Prominent bands were observed around
found similar with the standard caffeine
(cm-1) 3107, 2943, 1720, 1253/1025, 1632
D1 (Figure 1).
and 1656 corresponding to =C-H, C-H,
C=O, C-N, C=C and C=N, respectively
(Figure 2). The IR spectrum of 1-13B
showed similar absorption bands with that
of the literature (Yang et al, 2012).

Figure 1.TLC of caffeine isolated from Tea and Coffee

70

65
Transmitance (%)

60
3107 1025

55 1253

50
2943

45
1632
40 1720
1656
35
4000 3000 2000 1000 0 5
Wave number (cm-1)

Figure 2. IR spectrum of caffeine isolated from black lion tea

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 Abebe and Gashu, BIRJSH, 2023, 7(1), 1–16

The 1H NMR spectrum contained three is more deshielded than C-2 due to the
singlet signals at δ3.35, 3.53 and 3.95 ppm presence of the double bond at position 5
assignable to three methyl protons at 1’, 3’ which makes the carbon more
1
and 7’ (Table 2, structure 1). In the H electronegative arising from the
NMR spectrum, the peak at δ7.49 was delocalization of electrons either at
observed due to the olefinic proton at C-8. position 3 or 9. C-5 is bonded to N-7 and
The deshieldings observed in the spectrum the carbonyl group, C6=O; whereas C-4 is
are mainly due to the nitrogen bonded to bonded to two nitrogen atoms N-3 and N-
13
the hydrogens [23]. According to C- 9. Thus, the bridging carbon signals of
NMR and DEPT spectra of 1-13B, there both C-4 and C-5 are expected to be
are eight carbon signals. Signals at δ27.86, deshielded, but C4 is more deshielded
29.68 and 33.53 represented methyl mainly due to the two nitrogen atoms
carbons bonded to nitrogen at positions 1, attached to it, hence the lone pair of
3 and 7. Carbon signals at δ107.52 (q), electrons on the nitrogen at position 3 and
141.41 (t), 148.64 (q), and δ151.65 (q) and 9 increases the resonance structure. The
δ155.34 (q) were ascribed to C-5, C-8, C- signal at δ 141.41 is mainly due to the
4, C-2 and C-6, respectively. Signals at presence of the double bond (C-8) (Yang
position 2 and 6 are highly deshielded. C-6 et al, 2012; Sitkowski et al, 1995).
Table 2 .1H and 13C NMR spectral data of 1-13B compared with caffeine

C NMR data of 1-13B NMR data of caffeine

1 13 1 13
no H-NMR C-NMR H-NMR C-NMR

1 δ 3.35 δ 27.86 δ 3.37 δ 27.5

2 δ 151.65 δ 151.3

3 δ 3.53 δ 29.68 δ 3.55 δ 29.3

4 δ 148.64 δ 148.3

5 δ 107.52 δ 107.1

6 δ 155.34 δ 154.9

7 δ 3.95 δ 33.53 δ 4.01 δ 33.2 6

8 δ 7.49 δ 141.41 δ 7.58 δ 141.2

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 Abebe and Gashu, BIRJSH, 2023, 7(1), 1–16

The COSY (1H-1H correlation The COSY spectrum shows no any proton-
spectroscopy), HSQC (Heteronuclear proton coupling and HSQC shows the
Single Quantum Correlation) and HMBC proton signals at position 1’, 3’, 7’ and 8
(Heteronuclear Multiple Bond Correlation) correlated with the carbon signals at
experiments gives information on proton- position 1, 3, 7 and 8 respectively. From
proton coupling, correlations of the HMBC spectra, the N-methyl group at
chemical shift of proton with the chemical position 1 correlate with C-2 and C-6, the
shift of the directly bonded carbon and N- methyl group at position 3 correlates
utilizes multiple-bond couplings over two with C-2 and C-4 and the N- methyl group
or three bonds (J=2-15Hz) (Yang et al, at position 7 correlate with C-5 and C-8
2012; Sitkowski et al, 1995). (Table 3).

Table 3. COSY and HSQC NMR data of 1-13B

Carbon no. NMR data of 1-13B

COSY (1H-1H) HSQC (1H-13C)
correlation
1 H-1 (δ3.35)--------C-1
(δ27.86)
3 No correlation H-3 (3.53)---------C-3 O CH3
H3C 6
(29.68) 5 N7
1N
8
2
N 4 N
O 3 9
CH3

7 H-7 (3.95)--------C-7 Key HMBC correlations

(33.53)
8 H-8 (7.49)--------C-8
(141.41)

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Based on spectroscopic analysis and components for coffee quality and human
literature reports, compound 1-13B/1-10B health problems if taken in excess, was
was identified as caffeine (1) (Sitkowski et analyzed using UV-Vis in tea and coffee
al, 1995). samples as follows.

1' O CH3 7' Calibration curve

H3C 6
5 N7 The absorbance of standard caffeine in the
1N
8
calibration curve gave a linear line at 274
2 N 4 N
O 3 9
nm over the range (r2 = 0.9992; n = 6)
3' CH3
with regression equation y = 0.004x +
1
0.153 [y = absorbance, x = concentration
mg/L] as shown in Figure 3.
Determination of caffeine in Tea and
coffee extracts using UV-Vis
spectroscopy
Based on the focus of this study, caffeine
level, as one of the determinant

0.7
y= 0.004x+0.153

2
0.6 R = 0.9992
Absorbance

0.5

0.4

0.3

0.2
20 40 60 80 100 120

Caffeine conc.(mg/L)

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Parameter Value Error results in Table 4 shows that samples 1-

--------------------------------------------------- 13B and 1-10B contain 90.75 and 94.25
ppm of caffeine in the samples.
A 0.15333 0.00642
Table 4. Caffeine concentration in tea and
coffee samples
B 0.00414 8.24786E-5
--------------------------------------------------- S.No. Sample Caffeine
--------- concentration
R SD N P (ppm)
--------------------------------------------------- 1 1-13B 90.75
0.99921 0.0069 6 <0.0001 2 1-10B 94.25
---------------------------------------------------
Figure 3. Calibration curve for
Validations method
standard caffeine concentration
In order to express the precision under the
Similarly, the same wavelength was used same operating conditions over a short
for the determination of absorbance of interval of time, repeatability and
caffeine in the extracts and in turn the reproducibility of caffeine analysis was
concentration of caffeine in the test determined by intra-and inter-day assay
extracts was determined from the and expressed as %RSD. The intra- and
calibration curve. The mean value (± S.D) inter-day analysis variation of the method
of slope and intercept were 0.004 ±8.247E- was investigated by analyzing six different
5
and 0.153 ±0.0064, +respectively. The concentrations of caffeine performed three
response linearity was ensured by the times a day for three days. The %RS D
coefficient factor 0.9992. In the range of ranged from 0.33 to 1.23 for intra-day and
0.24 – 0.66 mg/mL caffeine concentration, 0.19 to 1.10 for inter-day variation was
a good linearity was observed, which is recorded and shows the method validity
very convenient for the determination of (Table 5).
caffeine in tea and coffee samples. The

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Table 5. Intra and inter-day precision of UV-Vis spectroscopic method

Amount in ppm Intra-day precision Inter-day precision

Mean area ± SD % RSD Mean area ± SD % RSD
20 0.21 ± 0.01 0.83 0.25 ± 0.02 1.10
40 0.27 ± 0.01 1.23 0.32 ± 0.01 0.72
60 0.39 ± 0.02 0.633 0.41 ± 0.03 0.47
80 0.47 ± 0.03 0.83 0.49 ± 0.01 0.27
100 0.54 ± 0.01 0.54 0.58 ± 0.02 0.19
120 0.66 ± 0.02 0.33 0.66 ± 0.02 0.54

The slope and intercept values, which was only sensitive to the standard
0.004±8.247E-5 and 0.153 ± 0.0064, show drug, streptomycin. The diameters of the
the validity of this method for growth inhibition areas were in the range
quantification of caffeine in tea and coffee of 6.67-13.33 mm for caffeine isolated
samples. These results are in agreement from tea and 7.33.-13.67mm for that of
with those reported elsewhere coffee. In addition, the highest inhibitory
(Wondimkun et al, 2016; Chalmers, 2016). effects were observed against Salmonella
It can be suggested that, this method is typhi, while no activity was seen against L.
fast, simple and reliable for the estimation monocytogen using both extracts. The
of caffeine in tea and coffee. MIC values obtained confirm the existence
of a significant activity against the
Antibacterial activity of isolated caffeine
bacterial strains tested in this study, with
Caffeine has been reported to be an
MIC values ranging from 125- 250 µg/ml
inhibitor of microorganism growth in
for tea and 125- 500 µg/ml for coffee. The
various food and food products. The result
standard drug streptomycin was active
showed that caffeine shows antibacterial
against all reference bacteria (zone of
activity against E.coli, S.aureus and
inhibition range: 13.2-17.3 mm; MIC 3.6
Salmonella thyphi but not L. monocytogen,
(Figure 4, Table 6 and 7).

10

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1-13B E.coli 1-13B Salmonella typhyi 1-13B S. aureus 1-13B L.

monocytogen

St= streptomycin, positive control

1-10B E. coli 1- 10B Salmonella typhi. 1-10B S. aureus 1-10B L.

monocytogen

Figure 4. Antibacterial activity of 1-13B, 1-10B and streptomycin.

In summary, caffeine is a potential their growth. The concentrations of

antimicrobial agent against different caffeine found in tea and coffee are
bacteria, and therefore, could be used in enough to warrant the antibacterial effect
foods as a natural preservative to control against tested bacteria.

11

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Table 6. Antibacterial activity of caffeine isolated from tea and coffee

Organism Diameter of zone of inhibition (mm)

Caffeine source Control
Tea Coffee Streptomyc Solven
Concentration Concentration (mg/ml) in, μg/ml t
(mg/ml)
100 50 25 12. 100 50 25 12.5 10
5

E. coli 11. 1o. 0 0 11 9.6 9 8.6 17.1 90

7 3 7

S. aureus 12. 11 8.3 6.6 12 9 7.3 0 13.3 90

7 3

Salmonella 13. 10. 8.6 9 10.3 10 13. 11.7 17.3 90

3 7 7 7

L. 0 0 0 0 0 0 0 0 17 90
monocytogen

Table 7. MIC values of caffeine isolated from tea and coffee

Microorganisms MIC values (µg/ml) Control,

Caffeine source Streptomycin

Tea Coffee

S .aureus 125 125 3.8

E .coli 250 125 3.8

Salmonella 125 500 3.6

12
L. monocytogen …. …… 3.7

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 Amare et al., BIRJSH, 2021, 5(1), 192-207

Conclusion Amamo, A. A. (2014). Coffee production

and marketing in Ethiopia. Eur J Bus
In this study, caffeine was isolated and
Manag, 6(37), 109-22.
characterized using spectroscopic data.
The caffeine content of tea and coffee
samples were also determined using UV- Atomssa, T., & Gholap, A. V. (2011).

Vis spectrometer as 90.75 ppm and 94.25 Characterization of caffeine and

ppm, respectively. The isolated caffeine determination of caffeine in tea leaves

showed antimicrobial activities against using uv-visible spectrometer. African

bacteria which suggests its use in food Journal of Pure and Applied Chemistry,

preservation. In the meanwhile, the 5(1), 1-8.

average intake of caffeine either in the

form of tea and brewed coffee cups per Belay, A. (2011). Some biochemical
day should be determined. compounds in coffee beans and methods
developed for their analysis. International
Conflict of interest Journal of the Physical Sciences, 6(28),
The authors declare that they have no 6373-6378.
known conflict of interest.

Bhoo-Pathy, N., Peeters, P. H., Uiterwaal,

C. S., Bueno-de-Mesquita, H. B., Bulgiba,
Conflict of Interest
A. M., Bech, B. H., ... & Van Gils, C. H.
The authors declare that they have no (2015). Coffee and tea consumption and
known conflict of interest. risk of pre-and postmenopausal breast
cancer in the European Prospective
Investigation into Cancer and Nutrition
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