See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/338335612

Quantification of caffeine content in coffee bean, pulp and leaves from

Wollega Zones of Ethiopia by high performance liquid chromatography

Article · January 2020

CITATIONS READS

5 6,657

2 authors, including:

Aman Dekebo
Adama Science and Technology University
75 PUBLICATIONS   824 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Synthesis of bioactive compounds View project

phytochemical analysis and Biological activities View project

All content following this page was uploaded by Aman Dekebo on 02 January 2020.

The user has requested enhancement of the downloaded file.

 Trends Phytochem. Res. 3(4) 2019 261-274

Trends in Phytochemical Research (TPR)

Journal Homepage: http://tpr.iau-shahrood.ac.ir

Original Research Article

Quantification of caffeine content in coffee bean, pulp and leaves from Wollega Zones
of Ethiopia by high performance liquid chromatography

Zerihun Asfew¹ and Aman Dekebo1*

¹Adama Science and Technology University, P.O. Box 1888, Adama, Ethiopia

ABSTRACT ARTICLE HISTORY

Caffeine is a stimulant alkaloid found in aerial parts of many hot beverages, Received: 03 May 2019
including coffee and tea. Due to its health impact, quantification of caffeine Revised: 19 October 2019
level in coffee is of paramount importance for consumers and traders, as well. Accepted: 28 November 2019
ePublished: 14 December 2019
Therefore, this study was designed to determine the caffeine content in coffee
beverage prepared from coffee beans, pulp and leaves using high performance
liquid chromatography coupled with a reverse phase C₈ column and UV-detector. KEYWORDS
In this study, caffeine sample was extracted from coffee beans, pulp and leaves Caffeine
with boiled distilled water followed by solvent partition with chloroform. The Coffee beans
extracted caffeine samples were analyzed alongside caffeine standard solutions Coffee leaves
over the concentration range of 5-25 μg/mL. For quantitative purposes, the Coffee pulp
standard caffeine gave an equation of Y=1270560x + 986903 (R² = 0.998) and HPLC
the retention time of 1.84 ± 0.01 min. In parallel to the standard caffeine solution,
peak area of caffeine contents in 20 μL extracted caffeine samples of 2.5 g coffee
powder in 100 mL of distilled water coffee beverage were registered. By using
the peak area, caffeine concentration in injected sample and its concentration
in the total sample solution was calculated. The percentage masses of caffeine
(w/w%) in the original coffee samples were 1.30 ± 0.11% for beans, 0.90 ±
0.11% for pulp and 0.65 ± 0.10% for leaves. These results of caffeine contents
in coffee beans in Wollega zone show high caffeine contents when compared
with previously reported coffee beans (1.01 ± 0.04-1.19 ± 0.02%) of other parts
of Ethiopia using UV/Vis. spectrophotometric technique.
© 2019 Islamic Azad University, Shahrood Branch Press, All rights reserved.

1. Introduction “Sidama”, “Teppi” and “Yirgacheffe”. Under each coffee

type, 2-5 different local types are known. Such high
Coffee is derived from the name of the province Keffa level of diversity is partly attributed to the presence of
where shepherds from Abyssinia/Ethiopia discovered the indigenous traditional production systems of coffee in
coffee plant in the 6th century (Ranheim and Halvorsen, the country (Boot, 2011). Coffee beans are the seeds of a
2005; Weldegebreal et al., 2017). It has become one shrub belonging to the botanical family Rubiaceae and
of the most widely consumed beverages throughout the genus Coffea (Clifford and Ramirez-Martinez, 1991).
the world due to its pleasant taste, aroma, stimulant The generic name covers over sixty different species of
effect and health benefits (Chen et al., 1998; Camargo, which only three, namely Coffea arabica, Coffea robusta
1999; Schenker et al., 2002; Aresta et al., 2005; Perrone and Coffea liberica have commercial values (Boot,
et al., 2008; Gebeyehu and Bikila, 2015). In Ethiopia, 2011). The two most important commercial species are
there are different coffee types recognized by their Coffea arabica and Coffea canephora, usually known
origin and quality and used as trade names including as Arabica and Robusta varieties, respectively (Martın
“Bebeka”, “Harar”, “Jimma”, “Kaffa”, “Wollega”, “Limmu”, et al., 1998; Boot, 2011). Arabica is considered to be a
Corresponding author: Aman Dekebo
Tel: +251-222110056; Fax: +251-222100075
E-mail address: amandekeb@gmail.com
262 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

higher quality bean, prized for its complex aroma and As caffeine contents of the coffee samples varied on the
flavors and is usually the most expensive one in the basis of geographical locations, there should be further
world market (Martın et al., 1998). Coffee has strong studies on different geographical and environmental
historical, cultural, social and economical importance. conditions that results these differences (Gebeyehu and
It is also the single most important tropical commodity Bikila, 2015). In the coffee plant, caffeine is present in all
traded worldwide, accounting for nearly half of total of its aerial parts. Caffeine biosynthesis takes place in the
exports of tropical products (Fujioka and Shibamoto, leaves and the outer part of the fruit. However, in aged
2008). The world’s largest importer of coffee is the leaves, the caffeine content is lower (Oestreich-Janzen,
EU, accounting for 66% of worldwide imports ca 4.0 2013). In the pulp tissues, light strongly stimulates the
million tonnes, in 2008 followed by the United States methylation step of caffeine synthesis. When the seed
(24%, 1.5 million tonnes) and Japan (7.0%, 423,602 inside the fruit starts growing, caffeine is translocated
tonnes) (Wondimkun et al., 2016). Most of the coffee in through the membranes and accumulates in the
the world market is produced by developing countries endosperm. Then, the final value is reached 8 months
(Yigzaw et al., 2007). Ethiopia is one of world top ten after the flowering stage (Oestreich-Janzen, 2013).
exporters of Coffea arabica and (first in Africa) leading Researchers from England and France have discovered
to its domestic consumption in the continent. About 12 that a tea made from coffee leaves involves even more
million Ethiopians make their living from coffee. While antioxidants and healthful compounds than either
most of the coffee plants cultivated in Ethiopia are regular tea or coffee. Since geographical location is
Coffee arabica (Fig. 1), there are, however, wide ranges known to affect the caffeine content, coffee species
of variability among coffee cultivars in the country. belonging to the same variety can have different
This variability in coffee beans has been attributed to amounts of caffeine level (Gebeyehu and Bikila, 2015;
variation in the soil, altitude and climate of the coffee Wondimkun et al., 2016). Due to the widespread
growing areas (Yigzaw et al., 2007). These factors are consumption of caffeine and its potential physiological
believed to considerably influence coffees characteristics and pharmacological effects, it is important for both
like chemical content, flavor or aroma (Gebeyehu and health professionals and consumers to know the exact
Bikila, 2015). Both species, Arabica and Robusta, are rich caffeine content in food. It is therefore important to
sources of biologically active compounds. The coffee precisely determine the caffeine content in different
beverage is rich in bioactive substances such as nicotinic coffee types, as a way to assess their caffeine contents
acid, trigonelline, quinolinic acid, tannic acid, pyrogallic in order to find a more precise relationship between the
acid and especially caffeine (Minamisawa et al., 2004). amounts of consumed caffeine and their physiological
Caffeine (1,3,7-trimethylxanthine) (Fig. 2) is an odorless effects (Demissie et al., 2016). Despite the wide use of
and slightly bitter material that can be naturally found in coffee in Ethiopia, there is the lack of extensive studies
the coffee and cocoa beans, tea leaves and other plant addressing the issues related to the caffeine content
species (Castro et al., 2010; Amare and Admassie, 2012). of coffee growing in Wollega, Ethiopia. Coffee pulps
It can be consumed in the form of coffee, tea, cocoa, and leaves were also used sometimes as a beverage
chocolate and energy drinks where is intentionally in different parts of Ethiopia after roasting, grinding
added. Moreover, it is considered to be as one of the most and adding to the boiled water. For example, lactating
commonly consumed behavioral active substances with mothers in some Ethiopian regions use these beverages
more than 80 percent of the world’s population daily of pulps and leaves with milk. Though there were
consuming caffeine (Carrillo and Benitez, 2000; Norton reports of caffeine contents in coffee bean in literature,
et al., 2011). It is stimulant of central nervous system, to the best of our knowledge, there were no reports on
cardiac muscle and respiratory system, diuretic delays comparison of caffeine contents of coffee beans, pulps
and fatigue. The human salivary level, which indicates and leaves.
the extent of absorption, peaks around 40 minutes Weldegebreal et al. (2017) reported a direct method
after caffeine consumption (Chou and Bell, 2007). Many of determination of caffeine in aqueous solution of
studies have attracted considerable attention due to the green coffee bean using FT-IR-ATR and fluorescence
antioxidant and anticancer properties as well as health spectrometry. In this regard, among the several
benefits of caffeine containing plants. However, in analytical techniques which have been developed for
excess amount, caffeine is known to cause a higher risk the determination of caffeine and the quality control of
of developing bone problems, including osteoporosis products containing caffeine, high performance liquid
problems in metal absorption and excretion, re chromatography (HPLC) is the method of choice (Grujić-
absorption processes in intestines and kidney resulting Letić et al, 2016; Abbood and Aldiab, 2017) whenever
in iron deficiency anemia (Shaker et al., 2010). A fatal the sample cannot easily be converted to the gas phase.
dose of caffeine has been reported to be more than Therefore, in this context, this article will highlight
10 g which is equivalent to about 170 mg/kg of body the concentration of caffeine in coffee beans, pulps
weight (Grujić-Letić et al., 2016). and leaves in some areas of Wollega zone, Ethiopia
The amount of caffeine in a cup of coffee can vary and compare their contents using HPLC technique.
greatly, depending on both intrinsic factors involving Additionally, in this study, we have focused on the
the species and/or its origin along with extrinsic factors determination and comparison of caffeine contents of
such as sampling localities and the method of brewing. coffee beans growing in Wollega zones.
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 263

CH3
O

H3C N
N

O N
N

CH3

Fig. 2. The chemical structure

of caffeine.

Fig. 1. Picture of the aerial parts

of coffee plant.

2. Experimental Coffee pulp and beans were collected and removing of

pulp from this bean was made at laboratory.
2.1. Sample collection In order to prepare representative data for Wollega
zone, three districts were selected purposely based
2.1.1. Description of the study areas on their selection by dweller (consumers, traders and
agricultural office of zones) of the areas from high to
The study was conducted in three zones of Oromia low selective features. Based on the above selection,
region Kelem Wollega, Western Wollega and East coffee samples were collected from Anfilo, Sayyo and
Wollega which are located in the west of the country. Gidami for Kelem Wollega, Nole, Gimbi (Gambela)
Dembi Dollo town is the capital and administrative and Begi for West Wollega and Sasiga, Harolimu and
centre of Kelem Wollega zone and 652 km far from Addis finally Nunukumba for East Wollega. 10-g portions of
Ababa. For West Wollega, Gimbi town is the capital and coffee samples were measured from each district. The
administrative centre of zone and 441 km from Addis measured 10-g portions of Anfilo, Sayyo and Gidami
Ababa. Nekemte town is the capital and administrative coffee samples were added together and roasted as
centre of East Wollega zone and found at 322 km Kelem Wollega coffee sample representative. Again
from Addis Ababa. The study area is located between Nole, Gimbi (Gambela) and Begi were added together
latitude 8º12′-10º03′ N and longitude 34º08′-36º10′ E. for West Wollega and Sasiga along with Harolimu
The zones are classified into three agro-climatic zones and Nunukumba for East Wollega coffee sample
locally known as Kola, Woinadega and Dega. These representatives. The coffee sample from these three
selected study areas have suitable afromontane rain zones may be representative of Wollega brand coffee
forest, altitudes ranging from 1,300 to 1,800 m above sample. In support to this data, some papers dealing
sea level which contain density of coffee, annual rainfall with the Ethiopian coffee, have stated that there is only
varies from 1000 to 2400 mm and a wide range of soil 2-5 local variability for one brand of coffee (Boot, 2011).
types (from acidic to slightly acidic with low availability
of phosphorous) where its fertility is maintained by 2.2 Chemicals
organic recycling and suitable temperature of 15-25 °C,
cool, shady environment of the forest of south western The main chemicals and reagents used in this experiment
highlands of Ethiopia (Beyene et al., 2015). Wollega zone were distilled water, anhydrous sodium carbonate
is one of most coffee cultivating areas of the country. (Na₂CO₃), HPLC grade chloroform (CHCl₃), diluted
NaOH (10%), anhydrous sodium sulphate (Na₂SO₄),
2.1.2. Study design and selection of coffee sample site acetone, anhydrous caffeine and HPLC grade methanol.
Among various solvents used to extract caffeine from
Data were collected in February 2016. All samples of raw coffee solution, chloroform was selected due to its
beans, pulp and leaves were collected from the similar best caffeine extracting ability (Islam et al., 2002).
areas at an altitude rage of 1300-1800 m above sea level.
264 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

2.3. Caffeine extraction The caffeine extracted from each coffee sample was
placed into 100 mL volumetric flask and diluted up to
Coffee beans, pulp and leaves were extracted from the mark with HPLC grade distilled water. The resultant
commercial Arabica variety. 30-g portions of each solution was sonicated for 5 min. The solution was
coffee beans, pulp and leaves were roasted using a then filtered using Whatman No. 1 filter paper. From
local coffee roasting machine. Each of the roasted the filtrate solution, 15 mL was diluted to 100 mL using
samples were ground and screened through 250 μm HPLC grade distilled water. Then, its peak was analyzed
sieves to get a uniform texture. In the next step, 2.5 g alongside with standard caffeine using HPLC.
of sample powder was measured and added into 100
mL beaker. Then, 50 mL of the boiled water was added 2.6. HPLC analysis of caffeine
over the coffee bean powder. The beaker containing
solution of coffee was put on hot plate and occasionally Caffeine in the filtered beverages of coffee samples was
stirred for 15 min. The coffee solution was then poured analyzed using column German (Zobax-SB-C8) HPLC
through cheesecloth into another beaker and pressed and detected by UV/Vis detector at the wavelength
out the solution. Thereafter, the extracted solution of 272 nm and quantified using a calibration graph.
was put aside. The coffee bean powder residue on The extracted caffeine was identified by comparing
the cheesecloth was washed twice with 25 mL of hot the retention times and spectral data with those of
water for 10 min. Then, the obtained extracts were authentic standards. All analyses were repeated three
combined. The final coffee beverage extracts was times. The relative peak areas were determined for three
filtered by vacuum suction filtration method to remove replicates of each dilute sample including standard
any insoluble solids and cooled at room temperature. caffeine. In chromatographic analysis, sample was
1.0 g of sodium carbonate (Na₂CO₃) was added to this purified before being injected into HPLC. Reverse phase
solution in order to remove some inorganic compounds HPLC column (Zobax-SB-C8) was used to determine
that can react with Na₂CO₃ (Alliance and Chan, 2013) the concentration of caffeine in coffee beverage drinks.
and then 25 mL of HPLC grade chloroform (CHCl₃) was Using the high performance liquid chromatography
added to the sample solution and the mixture was system made a fast and easy separation of caffeine from
vigorously swirled for 10 min and allowed to stand and any other substances in the extracted caffeine sample.
being separated into two layers; a dark aqueous top Standard solution of caffeine was prepared and injected
layer and a clear chloroform bottom layer. The organic into the HPLC. From the resulting chromatograms,
and aqueous layers were separated using a separatory measurements of retention time (tR) and peak areas
funnel. The above procedure was repeated three times were performed. In this chromatographic determination,
by adding 25 mL of chloroform to the aqueous layer. retention time (tR) was used as a qualitative measure,
5.0 mL of an aqueous NaOH (10%) solution was added whereas the peak area was used as quantitative
into the combined organic extracts to remove inorganic measure. A calibration curve for peak area against the
impurity. 1.0 g of Na₂SO₄, as a dehydrating agent, was concentration of the caffeine standards was employed
added to remove any trace water molecule and the to determine the concentration of caffeine in the coffee
resulting extract was then filtered. The organic layer beverages.
was concentrated using rotary evaporator and yielded
caffeine. The entire above step were repeated for both 2.7. HPLC conditions
coffee pulps and leaves. Qualitatively, the extracted
sample was checked by TLC, HPLC and UV/Vis.-based HPLC-UV analysis was performed on an Agilent liquid
spectroscopic methods. chromatograph system (HP 1220, Agilent, USA). A
Zobax-SB-C₈ reversed-phase packed column, German,
2.4. Quantitative determination of caffeine in extracted Agilent Technology (4.6 mm x 150 nm: 5 μm) column
samples was used throughout this study. The concentration of
caffeine was determined by using high performance
2.4.1. Preparation of stock standard and working liquid chromatography equipped with UV detector
solutions (HPLC-UV) set at 272 nm and the run time of 6 min at a
flow rate of 1 mL/min at room temperature. An isocratic
Caffeine stock standard solution (1000 ppm) was elution was used using HPLC grade methanol (100%)
prepared by dissolving 100 mg of caffeine standard with a total run time of 6 min.
in 80 mL of distilled water and sonicated for 10 min.
Then, the obtained solution was transferred to 100 mL 2.8. Statistical analysis
volumetric flask and filled to the mark with distilled
water. This stock solution was stored in a dark place at All measurements and analyses were carried out in
+4 °C for two days. From the prepared stock solution, triplicates. The results were expressed as mean ±
10 mL was transferred to 100 mL volumetric flask and standard error of three parallel replicates. Analysis of
volume was made up to the mark with distilled water to variance was performed by using one way ANOVA. The
make the working solution (100 μg/mL). results with p < 0.05 were regarded to be statistically
significant. Data were statistically analyzed using SPSS
2.5. Preparation of extracted sample solution multiple comparison Tukey HSD programs.
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 265

2.9. Preparation of calibration curve caffeine sample was drawn and registered (Table 1).
Construction of calibration curve was done by taking
Chromatogram of standard solutions (5, 10, 15, 20, standard caffeine solutions (5, 10, 15, 20, 25 μg/mL)
25 μg/mL) being prepared from the working standard from the corresponding chromatogram. Finally, caffeine
solution (100 μg/mL) was monitored. The concentration content of coffee beverage under test was calculated
versus peak area response was also registered (Table from the extracted chloroformic sample solutions of
1). The external standard calibration method was used. coffee beans, pulp and leaves using linear regression
Alongside this standard solution, chromatogram of equation obtained from drawn calibration curve (Eqn.
the unknown concentration chloroformic extracted 1).
Table 1
Peak area of caffeine standard and extracted samples.

Conc. of Accepted
Peak area of Amount
standard Part of Sample Peak area (units)2
standard injected precision
caffeine coffee area (μL) (Mean±SD) (n = 3)
(units) 2
RSD%
(μg/mL)
5 7,853,096 Kelem Wollega 20 14579975.7±249671.1 1.71
Beans West Wollega 20 13114625.7±113786.7 0.87
10 13,224,913 East Wollega 20 12516925.4±18969 1.5

Kelem Wollega 20 10226526.67±204407.1 1.99

Coffee
15 19,877,730 West Wollega 20 10226986.67±170008.8 1.66
pulp
East Wollega 20 8453430.7±144988.7 1.73

20 26,082,152 Kalem Wollega 20 8170453±123643.1 1.5

Coffee
West Wollega 20 7044954.3±14620.9 0.23
leaves
25 33,188,404 East Wollega 20 6426735.3±39894.4 0.67

(Average ± Standard deviation (n = 3).

2.10. Method validation 2.10.2. Precision

Validation is a process of establishing documented According to the ICH guidelines (Association;

evidence which provides a high degree of assurance Gowrisankar et al., 2010), precision should be performed
that a specific activity will consistently produce a at two different levels; repeatability and reproducibility.
desired result or product meeting its predetermined Repeatability is an indication of how easy it is for an
specifications and quality characteristics. Due to this operator in a laboratory to obtain the same result for
reason, precision, linearity, accuracy, sensitivity, limit the same batch of material using the same method at
of detection (LOD) and limit of quantification (LOQ) of different times using the same equipment and reagents
the technique used were checked on the sight of ICH in the same day with minimum of determinations
guidelines (Gowrisankar et al., 2010). covering the specified range of the procedure.
Reproducible precision results from variations such
2.10.1. Detector linearity as different days, analysts and equipment. Precision
criteria for an assay method are that the instrument
Five aliquots were prepared from the secondary stock precision and the intra-assay precision (RSD) will be ≤
solution 100 μg/mL ranging from 5-25 μg/mL and 2% (Gowrisankar et al., 2010).
analyzed according to the experimental conditions.
The caffeine solution was scanned using HPLC 2.10.2.1. Intra-day (repeatability) precision study
against mobile phase as blank. It was found that the
linearity of sample between the ranges of 5-25 μg/mL In intra-day variation studies, solutions of the same
showed acceptable linear regression coefficient (R2 = concentration (5 μg/mL) were analyzed six times
0.9983). The calibration graph was generated using using the same method at the same time using the
20 μL injection loops. Then, the calibration curve was same equipment and reagents in the same day. Then,
established according to the obtained response (peak sample peak area was measured by HPLC and the mean
area) and the concentrations of caffeine in standard standard deviation and RSD of sample were calculated
solutions. The results show a good linear relationship. (Table 2).
 266 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

2.10.2.2. Inter-day (reproducibility) precision study 2.10.3. Accuracy study

In the inter-day variation studies, solutions of the same The accuracy of the study was determined by percent
concentration (5 μg/mL) were analyzed three times for of recovery. For percent of recovery study, one sample
the two consecutive days by the same method at the of known caffeine concentration from different types of
same time using the same equipment and reagents coffee beverages was spiked with 5 mg/L of caffeine
and the sample peak area and the mean retention time, standard and recovery was calculated as summarized
standard deviation and RSD(%) were calculated (Table in (Table 1). All analyses were carried out in triplicate.
2). General equation used to calculate the recovery (%) is
given in equation below (APHA, 1999).
A. intra-day precision study of 5 μg/mL standard caffeine peak area and retention time; B. inter-day precision of 5 μg/mL caffeine

Precision measuring parameter

RSD%
RSD%

1.28
0.36
1.15

0.36

Precision measure pa-

2.10.4. Limit of detection (LOD) and limit of

quantification (LOQ)
rameter

772009.5 9937.76
0.0067
8961.4

0.0066

The limit of detection (LOD) is defined as the lowest

SD

concentration of an analyte in a sample that can

SD

be detected but not quantified. It is expressed as a

concentration at a specified signal:noise ratio usually as
775889
Mean

Mean

3:1. The LOQ signal:noise ratio of 10:1 is defined as the

1.84

1.84

lowest concentration of an analyte in a sample that can

be determined with acceptable precision and accuracy
under the stated operational conditions of the method.
783576

1.843

As ICH has recommended, LOD and LOQ may also be

calculated based on the standard deviation (SD) of the
6

response and the slope of the calibration curve(s) at

774231
782745

1.857

levels approximating the LOD according to the formula

6
1.847

given below (Gowrisankar et al., 2010).

5

No of replicates
06/07/2016

776897
762318

1.843
5
1.837
4

Where Sa is the standard deviation of residue and b the

slope of the calibration line, respectively.
No of replicates

754231
767542

However, in order to calculate LOD and LOQ, the

1.847
4
1.857

value of standard deviation of residue and intercept

were calculated by microsoft office excel windows 10
3

program from calibration curve of the standard caffeine

Sa = 245549.8374 and Sb = 1288737.953.
776897 767542
776897

1.843
3
1.843

3. Results and Discussion

peak area and retention time at different day.

3.1. Ethnomedicinal values of some plants in the study

No of replicates

1.843
2

area
05/07/2016

The study areas in Wollega region have several

traditional medicinal plants which belong to families
782259

782259

including Asteraceae, Solanaceae, Zygophyllaceae,

Retention time 1.847

Retention time 1.837

1

Fabaceae, Rosaceae, Musaceae, Euphorbiaceae,

Rutaceae, Malvaceae, Brassicaceae and Cucurbitaceae
1

(Feyissa et al., 2017). (Sarker and Nahar 2018) reviewed

phytochemicals which are natural antioxidants such as
Parameter

Parameter

curcumin, resveratrol, epicatechin, ellagic acid, flavone

Peak area

Peak area

and apigenin (Ng et al., 2000; Reuter et al., 2010;

Ribeiro et al., 2015) and are used in cosmetic industries
Table 2

to prepare various anti-ageing products. Additionally,

A

B
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 267

many phytochemicals such as phenolic compounds various medicinal plants fairly distributed throughout
and mono- and sesqui-terpenes or phyto-extracts the region (Feyissa et al., 2017). The identified plants
including Castanea sativa are also added to skin- in the region have a broad spectrum of activities and
care cosmetic products because of their antimicrobial are used for the treatment of multiple ailments and
properties (Ribeiro et al., 2015). Wansi et al. (2018 and have medical value against many diseases. (Feyissa et
2019) have reviewed several essential oils extracted al. 2017) reported that Citrus aurantifolia is among the
from various plant parts, such as leaves, bark, fruit, traditional medicinal plants available in this study area
roots and rhizomes which exhibited bioactivities having insecticidal property against lice infestation.
against Plasmodium falciparum, food borne microbes, Additionally, C. aurantifolia is considered as tonic for
dermatophytes, the malaria vector Anopheles gambiae, libido and as antidote for poison. The diluted form of
cancer cell lines, river blindness as well as plant the C. aurantifolia fruit juice is used for mouth wash
pathogen weevils and fungi. The Achillea species were to treat sore mouth, sore throat and is useful to treat
reported to have tonic, sedative, diuretic, carminative irritation, diarrhea and swelling due to mosquito bites
remedies which promote breast-feedings and regulate (Aibinu et al., 2007; Khare, 2007; Akhtar, 2013).
women menstruation and are extensively prescribed
for the treatment of stomachache, inflammation, 3.2. Detection of caffeine from coffee bean, pulp and
gastrointestinal, hemorrhoid, hay fever and wound leaves
healing in indigenous medicines (Mohammadhosseini
et al., 2017). Quantification of caffeine content in the test samples
(Abera 2014) reviewed the majority of medicinal plant was performed by an HPLC instrument coupled with
parts in Gimbi, Wollega zone, Ethiopia which are C8 column and UV-detector at the wavelength of 272
prepared either in combination with other medicinal nm. Fig.s 3-4 show general features of standard and
plant parts or with other additives such as boiled coffee, extracted caffeine chromatograms. The relative peak
honey and local beverages (tella) for different purposes areas of standard caffeine and three replicates of each
either to increase the healing potential or to improve diluted chloroformic extraction of coffee bean, pulp and
the flavour and taste or to avoid abdominal discomfort leaf samples were shown in (Table 1). From standard
(Tamene, 2000; Balemie et al., 2004). For instance, caffeine concentration prepared over the range 5-25
a traditional medicine applied to treat tape worm µg ̸ mL, linear regression calibration curves were made
infection is prepared by the combination of several (Fig. 5). Before HPLC analysis of the standard samples,
medicinal plant parts, i.e. Hagenia abyssinica, Glinus we checked the volume of the sample which gives the
lotoides with other additives such as local beverages best peak. Accordingly, 20 µL injection volume was
and salt (Balemie et al., 2004). There were also different found to be the best for the analysis. Regarding the
studies that showed the presence of a wide range of similar studies of contents of caffeine analysis, 20 µL
herbal medicines in the Wollega zone being used was selected as an optimal injection volume in such
for treating various ailments due to the presence of sorts of HPLC-based determinations (Ali et al. 2012).

Fig. 3. HPLC chromatogram of caffeine extracted from coffee beans, pulp and leaves(a: Kellem Wollega; b: West
Wollega; c: East Wollega).
268 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

Fig. 4. HPLC Chromatogram of caffeine standard sample.

Fig. 5. Calibration curve of standard caffeine.

3.3. Method validation to be not significantly different from the value of added
caffeine concentration. The result of accuracy (Table 3)
The method was validated in terms of linearity range, was within the range of ICH guideline. The accuracy(%)
intra-day precision (Table 2), inter-day precision (Table indicated non-interference from the component of
2) and analytical recovery and accuracy (Table 3), limit of solution. The results of analysis of beans were good and
detection and limit of quantification (Table 3). Generally, shown in Table 3.
the obtained calibration curve was found to be linear
over the concentration range of 5-25 μg/mL with an 3.4. Detection and quantification limits
acceptable correlation coefficient (R²) and a linear
regression equation used to calculate concentration of In Table 3, the calculated LOD and LOQ have been
caffeine in the extracted sample. From the quantitative shown using microsoft office excel windows 10. Hence,
analysis, acceptable relative standard deviation of 1.15% the lowest concentration that can be quantified with an
and 1.28% with stable retention time 1.84 ± 0.0066 min acceptable accuracy and precision (LOQ) of extracted
were resulted. Therefore, Table 2 represents the intra- caffeine samples and lowest concentration that can be
and inter-day precision of the new method, confirming detected but cannot be quantified (LOD) were given
adequate sample stability and method reliability over a in Table 3. The chromatograms of extracted caffeine
24 h period. samples obtained from coffee beans, pulp and leaves
The mean recoveries of the obtained results were found were shown in Fig. 3.
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 269

Table 3
Method detection and quantification limits of measured caffeine.

Standard deviation residue slope of the calibration line Calculated LOD Calculated LOQ
Sa=245549.8374 b=1288737.953 0.63 µg/mL 1.90 µg/mL
General formulae used to calculate
3.5. Linearity Using the regression equation (Eqn. 3) of standard
caffeine, the caffeine concentration of the extracted
A linear regression of peak area versus standard caffeine sample solutions were calculated as shown in Table 4.
concentration gave Eqn. 3 which is used to determine A linear regression concentration calculated from peak
the concentration of unknown chloroformic extracted area of injected chloroformic caffeine sample allowed
caffeine sample. to calculate the total concentration of caffeine (Eqn. 4)
in the extracted sample solution (Table 4).
y = 1270560x + 986903 (Eqn. 3)

Table 4
Caffeine concentration of beans, pulp and leaves of extracted samples.

Caffeine
Caffeine Caffeine Conc. Caffeine to
Caffeine content Total
Part of Sample Conc. in in concentrated coffee
coffee diluted sample Content (mg/100 average
area Ratio.
plant Sample (μg/ mL)
mL) (μg/mL) (mg) (Wt/Wt%)
(Wt/Wt%)

Kelem Wollega 10.69 ± 0.19 71.34 ± 1.30 35.67 ± 1.30 35.67 ± 1.30 1.43 ± 0.19
Beans
West Wollega 9.54 ± 0.085 63.55 ± 0.43 31.81 ± 0.43 31.81 ± 0.43 1.27 ± 0.08
1.30 ± 0.11
East Wollega 9.08 ± 0.14 60.58±0.97 30.29±0.97 30.29±0.97 1.21 ± 0.14

Kelem Wollega 7.24 ± 0.13 48.28 ± 0.92 24.14 ± 0.81 24.14 ± 0.81 0.96 ± 0.13

Coffee
West Wollega 7.25 ± 0.16 48.33 ± 0.93 24.17 ± 0.87 24.17 ± 0.87 0.97 ± 0.16
pulp 0.90 ± 0.11
East Wollega 5.88 ± 0.11 39.15 ± 0.76 19.57 ± 0.76 19.57 ± 0.76 0.78 ± 0.11

Kelem Wollega 5.68 ± 0.05 37.80 ± 0.34 18.90 ± 0.34 18.90 ± 0.34 0.76 ± 0.05
Coffee
West Wollega 4.76 ± 0.02 31.71 ± 0.17 15.85 ± 0.17 15.85 ± 0.17 0.63 ± 0.02
leaves 0.65 ± 0.10
East Wollega 4.29 ± 0.03 28.53 ± 0.20 14.26 ± 0.20 14.26 ± 0.20 0.57 ± 0.03

Average ± Standard deviation (n = 3)

Caffeine in total sample (ppm) = Conc. in injected (ppm) × dilution factor (Eqn. 4)

The final caffeine content in mass unit (mg) of the beverage under test was then calculated from the extracted
sample solution concentration using Eqn. 5 and Eqn. 6.
2

Caffeine content in injected sample (mg)= (Eqn. 5)

(Wondimkun et al., 2016)

Caffeine content in total sample (mg) = Caffeine content in injected sample (mg) x dilution factor (Eqn. 6)
The final result of the study was reported in terms pulps and leaves. Caffeine content of coffee beans
of percentage mass of calculated caffeine to total ranged from 1.21 ± 0.14% to 1.43 ± 0.19%, caffeine
mass of original sample used (W/W%). The yield of content of coffee pulp was within range of 0.78 ± 0.11%-
isolated caffeine from the collected samples of coffee 0.97 ± 0.16% and that of coffee leaves was within range
is presented in Table 4. The results indicate that coffee of 0.57 ± 0.03%-0.76 ± 0.05%. This result of the study
beans contain higher amounts of caffeine than coffee can be graphically shown in Fig. 6.
270 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

Fig. 6. Graphical representation of determined caffeine %Wt/Wt of

coffee beans, pulp and leaves collected from study area (0.5-1.5%).
KWseed: Kelem Wollega coffee bean; WWseed: West Wollega bean,
EWseed: East Wollega bean; KWpulp: Kelem Wollega pulp; WWpulp:
West Wollega pulp; EWpulp: East Wollega pulp; KWleaf: Kelem Wollega
leaf; WWleaf: West Wollega leaf; EWleaf: East Wollega leaf. Meanseed:
mean of bean; Meanpulp: mean of pulp and Meanleaf: mean of leaf.

3.6. Comparison of caffeine content in different parts (Oestreich-Janzen, 2013).

of the plants and among the regions In support to this study, different caffeine contents
in coffee beans have been reported by the previous
As shown in Fig. 3, the caffeine level of coffee beans, researchers. For instance, average caffeine content of
pulp and leaves determined in the study areas were coffee beans (1-2.2%) (Komes et al., 2009; Ali et al., 2012)
decreased in the order of coffee beans, coffee pulp were found to be over the range of 0.96 ± 0.01%-1.23
and coffee leaves. Within each coffee bean, the caffeine ± 0.06% for Arabic green coffee beans (Gebeyehu and
content decreases in order of Kelem Wollega (1.43 ± Bikila, 2015) together with 0.8-1.4% for Coffea arabica
0.19%), West Wollega (1.27 ± 0.08%) and East Wollega (Belitz et al., 2009) and less than 1.5% for Coffea arabica
(1.21 ± 0.14%). When caffeine content of coffee pulp (Illy, 2013). Moreover, the caffeine content in Ethiopian
is considered, it decreases in order of West Wollega Coffea arabica grown in Bench Maji, Gediyo Yirgachefe,
(0.97 ± 0.16%), Kelem Wollega (0.96±0.13%) and East Tepi and Godere which has been determined by UV/Vis
Wollega (0.78 ± 0.11%). For coffee leaves, order of their spectrophotometry to be 1.1 ± 0.01%, 1.01 ± 0.04%,
caffeine content decreases from Kelem Wollega (0.76 ± 1.07 ± 0.02% and 1.19 ± 0.02%, respectively (Belay
0.05%), West Wollega (0.63 ± 0.02 %) and East Wollega et al., 2008) accounting for less caffeine content than
(0.57 ± 0.03%) (Table 4). the coffee beans of studied area. On the other hand,
The result of our analysis on caffeine content in the caffeine content in Ethiopia and Eritrea Coffea arabica
studied area is in a good agreement with previously grown in Gojjam area, Geisha, Harar and Eritrea has
reported Coffea arabica beans caffeine content (Illy, been determined by HPLC technique to be 0.89-1.04%,
2013) which state less than 1.5% for Coffea arabica 0.93%, 1.18% and 0.95%, respectively (Gebeyehu
variety. In case of coffee leaves, caffeine content of the and Bikila, 2015). In general, comparison of previous
studied area was within a range of reported literature literature reports with this study for Coffea arabica
for Coffea arabica variety range between 0.62 to 0.98% indicates Wollega Coffea arabica has greater caffeine
(Oestreich-Janzen, 2013). For coffee pulps, caffeine content than other parts of Ethiopian Coffea arabica.
content obtained was between ranges of Coffea species Based on the previous literature reports, there may
caffeine content (0.54-1.67%) stated in literature be slightly variation of roasting condition (Illy, 2013)
(Clifford and Ramirez-Martinez, 1991). According to since roasting does not significantly affect the content
this study, quantity of caffeine present in all aerial of caffeine. Other factors like brewing strength,
parts of coffee plant (beans, pulp and leaves) have grinding degree, brewing methods and serving size
good agreement with previously reported papers in were controlled under the experimental conditions.
literature that state in coffee plant, caffeine is present Therefore, the variation in caffeine level of coffee
in all parts of the plants (Oestreich-Janzen, 2013). This samples may be due to geographical origins which
fact consistent with the literature report which shows might have different altitude, soil type, rain fall and other
caffeine biosynthesis takes place in the leaves and the agricultural conditions, e.g. harvesting time, exposed to
outer part of the fruit but when the seed inside the sun etc. as well as environmental conditions. In supports
fruit starts growing, caffeine is translocated through to this reason, other researchers reported that suitable
the membranes and accumulates in the endosperm environmental condition improves photosynthesis
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 271

and increases leaves area index, resulting in better other agricultural as well as environmental
producing of larger and heavier fruits with better beans conditions.
quality (Gole, 2003; Bote and Struik, 2011; Gebeyehu On the other hand, when brewing strength of
and Bikila, 2015; Wondimkun et al., 2016). Therefore, this study, 25 g/L coffee beverage was explained
greater caffeine content of Coffea arabica grown in in terms of 70 g/L ISO common brewing strength
Wollega zones with suitable afromontane rain forest, (Oestreich-Janzen, 2013) of coffee beverage and
altitudes range, annual rainfall and a wide range of the caffeine contents of Kelem Wollega, West
soil types (Gole, 2003) may be due to this suitable Wollega and East Wollega coffee beans were
environmental condition. respectively 99.88, 89.26, 84.81 mg and in good
On the other hand, as ANOVA result indicates caffeine agreement with previously reported data in
content of coffee grown in Wollega zones determined literature which state that on the average, a cup
from beans in Kelem Wollega, West Wollega and of coffee contains 80 mg to 175 mg of caffeine
East Wollega shows variation. The level of statistical depending on what "bean" (seed) is used and
significance association between the level of caffeine how it is prepared (Juliano and Griffiths, 2004).
content in beans, pulp and leaves were statistically
significant (p < 0.05) except for Kelem Wollega and 4. Concluding remarks
West Wollega coffee pulps which are insignificant. The
major source of this variation may be due to quality Following extraction of caffeine from coffee
of coffee seed originated from different agricultural and quantification by chromatographic HPLC
system and harvested time. analysis using peak area of extracted sample
In case of caffeine content, difference among Kelem alongside standard caffeine calibration curve,
Wollega, West Wollega and East Wollega coffee leaves the following results were reported in % mass of
the main source of difference may be originated from determined caffeine to its original coffee sample.
total caffeine content of original coffee from where The obtained results of this study are in good
they are collected. In support to this idea, experimental agreement with previously reported caffeine
results of this study show greater caffeine contents of contents of Coffea arabica even though it has a
Kelem Wollega coffee beans which have in turn greater greater value than those reported for the same
caffeine content in case of thier leaves, as well. Coffee coffee beans in some parts of Ethiopia. Although
leaves of East Wollega have lower caffeine content the number of coffee samples analyzed here is
which corresponds to less caffeine containing coffee still small, the data presented in this study gave
beans according to this report. On other hand, even a representative data about the caffeine content
though all this coffee leaves are collected at similar level of coffee plant that grows in Wollega zone.
season, agro-climatic difference may cause different Even though coffee pulps and leaves are used
maturation stages that causes variation of caffeine in rarely as beverage substituting coffee beans
leaves. This fact is briefly stated in literature ((Illy, 2013; in different parts of Ethiopia, most of the time,
Oestreich-Janzen, 2013). Additionally, for caffeine these parts of the plants are considered as by-
content variation of Kelem Wollega, west Wollega and products of coffee plants. Our results of caffeine
East Wollega coffee pulp, the main reasonable origin of contents of these parts of the plant may be of
the variation may be beans quality of caffeine content. interest for researchers interested to work further
In summary, in spite of the fact that the samples were in the preparation of beverages of coffee pulps
analyzed under similar experimental condition and and leaves with lower costs compared with those
relatively similar geographical location, variation of of coffee beans.
caffeine content within each coffee beans from the
three study areas could be due to harvesting time, Conflict of interest
agricultural system as well as environmental conditions.
For instance, as stated in literature (Bote and Struik, The authors declare that there is no conflict of
2011), coffee plants grown under shade trees produce interest.
larger and heavier fruits with better beans quality than
those grown in direct sunlight. Accordingly, the coffee Acknowledgements
sample growing around Kelem Wollega under shaded
area compared to those located in West Wollega and We acknowledge the Wollega zones Agricultural
East Wollega has different quality even though Wollega Office for supplying the author with coffee
zone belongs to rain forest part of Ethiopia. This fact is samples and background information on coffee
supported by experimental result which shows Kelem this area.
Wollega coffee has high caffeine content in all parts
of coffee plant except caffeine content of coffee pulp References
which is less than west Wollega coffee pulp. Taking
into account the above explanation, caffeine content Abera, B., 2014. Medicinal plants used
variation between Coffea arabica samples in different in traditional medicine by Oromo people,
parts of Ethiopia is due to geographical origins which
Ghimbi District, Southwest Ethiopia. J.
might have different altitude, soil type, rain fall and
272 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

Ethnobiol Ethnomed. 10(1), 40. http://www. Coffee, tea, cocoa. Food Chem. 938-970.
ethnobiomed.com/content/10/1/40. Beyene, B., Tolosa, T., Rufael, T., Hailu, B.,
Abbood, A., Aldiab, D., 2017. HPLC Teklue, T., 2015. Foot and mouth disease
determination of caffeine in some beverages in selected districts of western Ethiopia:
and pharmaceutical dosage forms available in seroprevalence and associated risk factors. Rev.
Syrian market. J. Chem. Pharm. Sci. 10(3), 1174- Sci. Tech. Off. Int. Epiz 34(3), 2.
1179. Bote, A.D., Struik, P.C., 2011. Effects of shade
Aibinu, I., Adenipekun, T., Adelowotan, T., on growth, production and quality of coffee
Ogunsanya, T., Odugbemi, T., 2007. Evaluation (Coffea arabica) in Ethiopia. J. Hortic. For. 3(11),
of the antimicrobial properties of different parts 336-341.
of Citrus aurantifolia (lime fruit) as used locally. Carrillo, J.A., Benitez, J., 2000. Clinically
Afr. J. Tradit. Complement. Altern. Med. 4(2), significant pharmacokinetic interactions
185. between dietary caffeine and medications. Clin.
Akhtar, S., 2013. Evaluation of cardiovascular Pharmacokinet. 39(2), 127-153.
effects of Citrus aurantifolia (Linn.) fruit. Available Castro, J., Pregibon, T., Chumanov, K.,
at SSRN: https://ssrn.com/abstract=2279447. Marcus, R.K., 2010. Determination of catechins
Ali, M.M., Eisa, M., Taha, M.I., Zakaria, B.A., and caffeine in proposed green tea standard
Elbashir, A.A., 2012. Determination of caffeine in reference materials by liquid chromatography-
some Sudanese beverages by high performance particle beam/electron ionization mass
liquid chromatography. Pak. J. Nutr. 11(4), 336. spectrometry (LC-PB/EIMS). Talanta 82(5), 1687-
Alliance, C., Chan, S.C., 2013. Chemical Test 1695.
for Caffeine. Hong Kong Chemistry Olympiad Chen, Q.-c., Mou, S.-f., Hou, X.-p., Ni, Z.-m.,
for Secondary Schools (2013-14), Memorial 1998. Simultaneous determination of caffeine,
College. theobromine and theophylline in foods and
Amare, M., Admassie, S., 2012. Polymer pharmaceutical preparations by using ion
modified glassy carbon electrode for the chromatography. Anal. Chim. Acta 371(2-3),
electrochemical determination of caffeine in 287-296.
coffee. Talanta 93, 122-128. Chou, K.H., Bell, L., 2007. Caffeine content
American Public Health Association (APHA), of prepackaged national‐brand and private‐
1999. Standard Methods for the Examination of label carbonated beverages. J. Food Sci. 72(6),
Water and waste Water. Washington DC. 20th Ed. C337-C342.
Aresta, A., Palmisano, F., Zambonin, C.G., Clifford, M., Ramirez-Martinez, J., 1991.
2005. Simultaneous determination of caffeine, Tannins in wet-processed coffee beans and
theobromine, theophylline, paraxanthine and coffee pulp. Food Chem. 40(2), 191-200.
nicotine in human milk by liquid chromatography Demissie, E.G., Woyessa, G.W., Abebe, A.,
with diode array UV detection. Food Chem. 2016. UV/Vis spectrometer determination of
93(1), 177-181. caffeine in green coffee beans from Hararghe,
Anonynous, 1985. Standard Methods for Ethiopia, using beer-lambert's law and
the Examination of Water and Wastewater. integrated absorption coefficient techniques.
American Public Health Association, American Sci. Study Res. Chem. Chem. Eng. Biotechnol.
Water Works Association, Water Environment Food Ind. 17(2), 109.
Federation, Washington. Feyissa, T.F., Hailemariam, T.B., Regassa, T.,
Belay, A., Ture, K., Redi, M., Asfaw, A., 2008. Kergano, N.K., 2017. Ethnobotanical study of
Measurement of caffeine in coffee beans with ethnoveterinary plants in Kelem Wollega Zone,
UV/Vis spectrometer. Food Chem. 108(1), 310- Oromia Region, Ethiopia. J. Med. Plants Res.
315. 11(16), 307-317.
Balemie, K., Kelbessa, E., Asfaw, Z., 2004. Fujioka, K., Shibamoto, T., 2008. Chlorogenic
Indigenous medicinal plant utilization, acid and caffeine contents in various commercial
management and threats in Fentalle area, brewed coffees. Food Chem. 106(1), 217-221.
Eastern Shewa, Ethiopia. E. J. Biol. Sci. 3(1), 37- Grujić-Letić, N., Rakić, B., Šefer, E., Milanović,
58. M., Nikšić, M., Vujić, I., Milić, N., 2016. Quantitative
Belitz, H.-D., Grosch, W., Schieberle, P., 2009. determination of caffeine in different matrices.
 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 251-274 273

Maced. Pharm. Bull. 62(1) 77-84. coffee. CAFEA GmbH, Hamburg, Germany.
Khare, C., 2007. Indian Medicinal Plants: An Perrone, D., Donangelo, C.M., Farah, A.,
Illustrated Dictionary Springer-Verlag. Berlin pg, 2008. Fast simultaneous analysis of caffeine,
699-700. trigonelline, nicotinic acid and sucrose in coffee
Gebeyehu, B.T., Bikila, S.L., 2015. Determination by liquid chromatography-mass spectrometry.
of caffeine content and antioxidant activity of Food Chem. 110(4), 1030-1035.
coffee. Am. J. Appl. Chem. 3, 69-76. Ranheim, T., Halvorsen, B., 2005. Coffee
Gole, T.W., 2003. Vegetation of the Yayu consumption and human health-beneficial
forest in SW Ethiopia: impacts of human use or detrimental?-Mechanisms for effects of
and implications for in situ conservation of wild coffee consumption on different risk factors
Coffea arabica L. populations. Cuvillier. for cardiovascular disease and type 2 diabetes
Gowrisankar, D., Abbulu, K., Bala Souri, O., mellitus. Mol. Nutr. Food Res. 49(3), 274-284.
Sujana, K., 2010. Validation and calibration of Reuter, J., Merfort, I., Schempp, C.M., 2010.
analytical instruments. J. Biomed. Sci. Res. 2(2), Botanicals in dermatology. Am. J. Clin. Dermato.
89-99. 11(4), 247-267.
Illy, E., 2013. The complexity of coffee. Sci. Ribeiro, A., Estanqueiro, M., Oliveira, M., Sousa
Am. 86-91. Lobo, J., 2015. Main benefits and applicability of
Islam, M., Rahman, M., Abedin, M., 2002. plant extracts in skin care products. Cosmetics
Isolation of caffeine from commercially available 2(2), 48-65.
available tea and tea waste. Jahangirnagar Uni. Sarker, S.D., Nahar, L., 2018. Phytochemicals
J. Sci. 25, 9. and phyto-extracts in cosmetics. Trends
Juliano, L.M., Griffiths, R.R., 2004. A critical Phytochem. Res. 2(4) 185-186.
review of caffeine withdrawal: empirical validation Schenker, S., Heinemann, C., Huber, M.,
of symptoms and signs, incidence, severity Pompizzi, R., Perren, R., Escher, R., 2002. Impact
and associated features. Psychopharmacology of roasting conditions on the formation of
176(1), 1-29. aroma compounds in coffee beans. J. Food Sci.
Komes, D., Horzic, D., Belscak, A., Kovacevic 67(1), 60-66.
Ganic, K., Bljak, A., 2009. Determination of Shaker, S.A., Farina, Y., Mahmmod, S., 2010.
caffeine content in tea and maté tea by using Synthesis and characterization of mixed ligand
different methods. Czech J. Food Sci. 27, 213- complexes of caffeine, adenine and thiocyanate
216. with some transition metal ions. Sains. Malays.
Martın, M.J., Pablos, F., González, A., 1998. 39(6), 957-962.
Characterization of green coffee varieties Tamene, B., 2000. A floristic analysis and
according to their metal content. Anal. Chim. ethnobotanical study of the semi-wetland of
Acta. 358(2), 177-183. Cheffa area South Wello. Unpublished MSc.
Minamisawa, M., Yoshida, S., Takai, N., 2004. Thesis, Addis Ababa University, Addis Ababa,
Determination of biologically active substances Ethiopia.
in roasted coffees using a diode-array HPLC Wansi, J.D., Sewald, N., Nahar, L., Martin, C.,
system. Anal. Sci. 20(2), 325-328. Sarker, S.D., 2018. Bioactive essential oils from
Mohammadhosseini, M., Sarker, S.D., the Cameroonian rain forest: A review - Part I.
Akbarzadeh, A., 2017. Chemical composition Trends Phytochem. Res. 2(4), 187-234.
of the essential oils and extracts of Achillea Wansi, J.D., Sewald, N., Nahar, L., Martin, C.,
species and their biological activities: A review. Sarker, S.D., 2019. Bioactive essential oils from
J. Ethnopharmacol. 199, 257-315. the Cameroonian rain forest: A review - Part II.
Ng, T., Liu, F., Wang, Z., 2000. Antioxidative Trends Phytochem. Res. 3(1), 3-52.
activity of natural products from plants. Life Sci. Weldegebreal, B., Redi-Abshiro, M.,
66(8), 709-723. Chandravanshi, B.S., 2017. Development of new
Norton, T.R., Lazev, A.B., Sullivan, M.J., 2011. analytical methods for the determination of
The “buzz” on caffeine: Patterns of caffeine use caffeine content in aqueous solution of green
in a convenience sample of college students. J. coffee beans. Chem. Cent. J. 11(1), 126.
Caffeine Res. 1(1), 35-40. Wondimkun, Z.T., Jebessa, A.G., Molloro, L.H.,
Oestreich-Janzen, S., 2013. Chemistry of Haile, T., 2016. The determination of caffeine
 274 Dekebo et al. / Trends in Phytochemical Research 3(4) 2019 261-274

level of wolaita zone, Ethiopia coffee using UV- bean caffeine, chlorogenic acids, sucrose and
visible spectrophotometer. Am. J. Appl. Chem. trigonelline contents among Ethiopian Arabica
4(2), 59-63. coffee accessions. SINET: Ethiop. J. Sci. 30, 77-
Yigzaw, D., Labuschagne, M., Osthoff, 82.
G., Herselman, L., 2007. Variation for green

View publication stats