DOI: 10.

1515/FV-2016-0026

FOLIA VETERINARIA, 60, 3: 34—38, 2016

STUDY OF ANTIOXIDANT EFFECTS OF SELECTED TYPES OF COFFEE

Hudáková, J., Marcinčáková, D., Legáth, J.

Department of Pharmacology and Toxicology

University of Veterinary Medicine and Pharmacy, Komenského 73, 04181 Košice
The Slovak Republic

dana.marcincakova@uvlf.sk

ABSTRACT substances were found also in extracts of roasted ground

100 % coffees Arabica and Robusta. The lowest levels of to-
Coffee is a rich source of dietary antioxidants which tal phenols (31.24 µg.ml–1) and flavonoids (0.91 µg.ml–1)
protects the human body against the effects of danger- were detected in the extract of instant coffee Arabica. The
ous free radicals. The aim of this study was to determine processing of coffee by roasting decreased the level of the
and compare the antioxidant activity, content of total investigated antioxidant components but considerably
phenols and flavonoids in selected types of coffee with improved the taste and aroma, the properties that make
respect to the way of their processing. The individual coffee one of the most popular drinks in the world.
coffees were investigated with regard to their origin and
composition. The antioxidant effects were determined Key words: antioxidant effect; Coffee Arabica (L.);
by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radi- free radicals
cal scavenging assay. The content of total phenols was
analysed by the Folin-Ciocalteu method and the content
of flavonoids in the coffee extracts was determined by INTRODUCTION
a colorimetric method. The highest antioxidant activity
was exhibited by the extract of unroasted ground 100 % The human body is constantly exposed to the effects
green coffee Arabica (89.55 %), and the high scavenging of free radicals produced by various metabolic pathways.
of free radicals was achieved also by the extracts of roast- They get into the bodies through air, liquids and food. They
ed ground 100 % coffees Arabica and Robusta. The high- are dangerous due to their ability: to attack cell structures,
est levels of total phenols (77.54 µg.ml–1) and flavonoids inhibit their normal functions, and thus contribute to the
(1.74 µg.ml–1) were measured in the extract of unroasted development of numerous diseases. Antioxidants play an
ground 100 % green coffee Arabica. High levels of these important role in the battle against the undesirable effects

34
of free radicals. The contribution of antioxidants is exten- by pouring boiling water (100 °C) over coffee samples and
sive; they protect human bodies against premature ageing, after 10 minutes, filtering the extracts and using them for
weakening of the immune system, and other health prob- the ultimate determination. All chemicals used in the ex-
lems caused by free radicals. The human body is capable periments were supplied by Sigma Aldrich (Germany).
of naturally producing its own effective antioxidants but
their amount is not always sufficient and thus it is neces-
Table 1. Coffee types used for the determination
sary to take them in the food. They are found, for example of antioxidant activity
in fruits, vegetables, chocolate, red wine and cereals. Re-
cently, increased attention has been paid to the beneficial Sample
Coffee type
No.
effect of coffee on human organism. Coffee is also an excel-
1 Arabica: roasted ground 100 % coffee (Gold)
lent source of antioxidants, particularly polyphenol com-
pounds, which participate considerably in the neutralisa- 2 Arabica: roasted ground 100 % coffee (Extra special)

tion of free radicals [9]. 3 Robusta: roasted ground 100 % coffee

Green (raw) coffee beans are the richest source of chlo-
4 Robusta: roasted ground coffee
rogenic acids, one of the strong antioxidants with a positive
5 Arabica: Instant 100 % coffee
influence on health. Research has demonstrated that these
acids can function as an inhibitor of glucose-6-phospha- 6 Arabica: Instant coffee (Crema Gold)
tase. Owing to chlorogenic acids, the body compensates 7 Arabica: unroasted ground 100 % green coffee
a decrease in the level of blood glucose by the break-down
of fats, and thus, green coffee is presently much sought-
after for the preparation of weight reduction. However, Spectrophotometric determination of antioxidant
chlorogenic acids are thermally unstable and thus undergo activity by the DPPH radical scavenging assay
many changes during roasting. Roasting is, however, an in- The antioxidant properties were determined by the
evitable way of processing of green coffee as it gives com- method of H e i l e r o v á et al. [6]. The principle consists in
plex aroma and taste to the final product (the properties the reaction of the coffee extract with a stable 2,2-diphe-
required by coffee consumers). The degree and conditions nyl-1-picryl-hydrazyl (DPPH). The ability of coffee extract
of roasting considerably affect the volatile components in to scavenge free radical is directly related to the rate and
coffee. Aroma of light roasted coffee differs considerably extent of decolouration of the synthetic radical DPPH and
from that of dark roasted coffee [10]. Owing to its pleasant at the same time as a decrease in the absorbance. The an-
aroma, taste and stimulating effect on the body and mind, tioxidant activity of coffee extracts was calculated as a per
coffee is one of the most popular drinks in the world. cent of inhibition of the DPPH radical. The procedure was
The aim of this study was to determine the antioxidant as follows: 3.9 ml aliquot of the stock solution of DPPH
properties and content of flavonoids and total phenols (0.0035 g per 100 ml methanol) was pipetted into a cuvette
in roasted and unroasted coffee varieties Coffea Robusta and the absorbance (A0) of this solution was measured at
and Coffea Arabica by means of the spectrophotometric 515 nm. Subsequently, we prepared a reaction mixture by
methods. adding 100 µl aliquot of coffee extract. After mixing and
incubation for 5 minutes, the absorbance of the reaction
mixture (AA) was measured. The antioxidant activity (per
MATERIALS AND METHODS cent of DPPH radical inhibition) was calculated according
to the formula: % inhibition = [(A0 – AA)/A0] × 100.
For analysis, we purchased from a store chain, 7 coffee
types (3 roasted and ground, 2 instant and one unroasted Spectrophotometric determination
green coffee) of varieties Coffea Robusta and Coffea Arabi- of the total phenols
ca. The list of coffee types is presented in Table 1. For prep- The content of the total phenols in the coffee extracts
aration of the extracts, we used 5 g quantities of ground was determined by the Folin-Ciocalteu method described
coffee and 2 g of instant coffee. The extracts were prepared in the study by S i n g l e t o n et al. [12]. The method is

35
based on the oxidation-reduction reaction during which 89.55 %) for scavenging DPPH radicals. High antioxidant
the phenol compounds are oxidised at a parallel reduction activity was determined also with extract of roasted ground
of Folin-Ciocalteu (FC) reagent and the development of 100 % Arabica — Sample 1 (82.5 %). The lowest percent-
a blue colour. The intensity of colouring correlates with the age of free radical scavenging was observed with extract of
redox properties of phenolic compounds present in coffee instant coffee with high content of coffee variety Arabica —
extracts. The procedure was as follows: coffee extracts and Sample 6 (56.16 %). Similar to antioxidant activity, the
the FC reagent were diluted with distilled water 1:1000 and extract of unroasted ground coffee exhibited the highest
1 : 10, respectively. Then, 1 ml of the diluted coffee extract content of total phenols (7754 µg.ml–1) followed by extract
was pipetted into a test tube and 5 ml of diluted FC-reagent of roasted ground 100 % Arabica — Sample 1 (73.64 µg.
and 4 ml Na2CO3 (75 g.l–1) were added and the content was ml–1). The level of total phenols was the lowest in Sample 6
mixed. After a 30 minute incubation at room temperature, (31.24 µg.ml–1); the extract of instant coffee with high con-
the absorbance of the solutions were measured at a wave- tent of coffee variety Arabica. The sample 7 rated the best
length of 765 nm, against a blank which contained distilled with regard to total flavonoids (1.74 µg.ml–1). The level of
water instead of the coffee extract. Gallic acid in concen- total flavonoids in the extracts of the remaining types of
trations of 0.038—0.3 mg.l–1 was used as a standard. Gallic coffee was above 1 µg.ml–1 with the exception of Sample 6,
acid absorbance ranges from 0.05 to 0.555 nm. instant coffee with high content of variety Arabica, where
it reached (0.91 µg.ml–1). The mean levels of the values and
Determination of total flavonoids standard deviations are presented in Table 2.
The content of total flavonoids was determined by the
colorimetric method described in the study by K i m et al.
[7]. The addition of an aluminium chloride solution to fla- DISCUSSION
vonoids results in the formation of yellow chelate complex-
es. The content of flavonoids was determined spectrophoto- Coffee is the main source of polyphenolic compounds
metrically by measuring the intensity of the yellow colour. which are well known for their antioxidant effects. They
The procedure was as follows: coffee extracts were diluted contain chlorogenic acids with biological effects mostly
with distilled water 1 : 100. To 1 ml of the diluted coffee ex- related to their remarkable antioxidant, anti-mutagenic,
tract, we added 4 ml of distilled water and 0.3 ml NaNO2 anti-carcinogenic and anti-inflammatory activities. The
(50 g.l–1). After 5 min of incubation, we added 0.3 ml AlCl3 chlorogenic acids exhibit a high capacity to scavenge reac-
(100 g.l–1), and after an additional 6 minutes, 2 ml NaOH tive oxygen radicals. These polyphenols are able to inhibit
(1 mol.l–1) and 2.4 ml distilled water, mixed the content and inflammatory processes and propagation of tumours by
measured the absorbance of the samples (510 nm) against means of the deactivation of pro-oxidative enzymes [5].
a blank which contained distilled water instead of coffee ex- Some studies demonstrated that green coffee extracts have;
tract. The content of total flavonoids was determined using anti-hypertension effects [8], inhibit the accumulation of
the quercetin standard (0.05—0.4 mg.l–1). fats and body weight gain [4] and modulate glucose metab-
olism in humans [1]. These biological effects were ascribed
Processing and presentation of the results to the chlorogenic acids present in green coffee.
The results of the determinations conducted in indi- The studies involved in the determination of the anti-
vidual coffee extracts are presented as arithmetic means oxidant capacity of coffee of a variety of Arabica showed
(x) and standard deviations (± SD). All measurements were that the content of total phenols in roasted coffee is lower
carried out in triplicate. than in unroasted green coffee [3], which correlates with
our results. Lower levels of polyphenols in roasted coffee
can be ascribed to their polymerization, autoxidation or
RESULTS degradation during roasting [2]. The content of total phe-
nols determined in our study correlates with the results of
Of all tested coffee types, the extract of unroasted C h e o n g et al. [3]. The highest antioxidant activity was
ground coffee exhibited the highest capacity (Sample 7; observed in the extract of green unroasted 100 % coffee

36
 Table 2. Antioxidant capacity, total phenols and flavonoids in the types of coffee (mean ± SD)

Antioxidant capacity Total phenols Flavonoids

Sample
[%] [µg.ml–1] [µg.ml–1]

1 82.5 ± 1.62 73.64 ±14.92 1.43 ± 0.18

2 78.92 ± 2.46 55.7 ± 6.11 1.05 ± 0.04

3 78.46 ± 0.66 51.37 ± 18.4 1.14 ± 0.08

4 79.93 ±1.49 59.9 ± 11.03 1.41 ± 0.05

5 71.06 ± 1.20 57.47 ± 11.24 1.02 ± 0.05

6 56.16 ± 4.09 31.24 ± 18.07 0.91 ± 0.01

7 89.55 ± 0.37 77.54 ± 15.36 1.74 ± 0.03

SD — standard deviation

Arabica. This coffee extract also showed the highest content ACKNOWLEDGEMENTS
of total phenols and flavonoids. The lowest antioxidant ca-
pacity was measured in the extract of 100 % coffee Robusta. This study was conducted with the support of National
Extracts of roasted ground coffees exhibited a comparable Reference Laboratory for Pesticides at UVMP in Košice.
capacity to scavenge free radicals. This capacity correlated
with the levels of total phenols and flavonoids. Relatively
lower levels of total phenols and flavonoids were recorded REFERENCES
in extracts of instant coffee. These samples exhibited also
lower antioxidant capacity. The results of our study agree 1. Blum, J., Lemaire, B., Lafay, S., 2007: Effect of a green
with the investigations of R a m a l a k s h m i and R a o [12] decaffeinated coffee extract on glycaemia. NutraFoods Research,
who reported that extracts of green coffee contain higher 6, 13—17.
levels of polyphenols and chlorogenic acids than extracts 2. Cammerer, B., Kroh, L. W., 2006: Antioxidant activity
of instant coffee. The relatively low levels of total phenols of coffee brews. European Food Research and Technology, 223,
can be explained by the degradation of these compounds 469—474.
during roasting and their release to water during extract 3. Cheong, M. W., Tong, K. H., Ong, J. J. M., Liu, S. Q., Cur-
preparation. ran, P., Yu, B., 2013: Volatile composition and antioxidant capac-
ity of Arabica coffee. Food Research International, 51, 388—396.
4. Dellalibera, O., Lemaire, B., Lafay, S., 2006: Le Svetol, un
CONCLUSIONS extrait de café vert décaféiné, inudit une perte de poids et aug-
mente le ratio masse maigre sur masse grasse chez volontaires en
The best antioxidant properties and the highest levels of surcharge pondérale. Phytothérapie, 4, 194—197.
total phenols and flavonoids were detected in ground green 5. Gawlik-Dziki, U., Świeca, M., Sułkowski, M., Dziki, D.,
coffee Arabica and the lowest in the extract of instant cof- Baraniak, B., Czyż, J., 2013: Antioxidant and anticancer activi-
fee with high content of variety Arabica. These results al- ties of Chenopodium quinoa leaves extracts–in vitro study. Food
lowed us to conclude that the antioxidant activity of coffee and Chemical Toxicology, 57, 154—160.
decreases with roasting, but coffee still retains considerable 6. Heilerová, Ľ., Bučková, M., Tarapčík, P., Šilhár, S., Labu-
capacity to scavenge free radicals and sufficient level of to- da, J., 2003: Comparison of antioxidant activity data for aqueous
tal phenols and flavonoids which also act as antioxidants. extracts of lemon balm (Melissa officinalis L.), oregano and agri-
mony (Agrimonia eupatoria L.) obtained by conventional meth-

37
ods and the DNA-based Biosensor. Czech Journal of Food Sciences, 11. Ramalakshmi, K., Rao, L. J. M., Takano-Ishikawa, Y.,
21, 78—84. 2009: Bioactivities of low-grade green coffee and spent coffee in
7. Kim, M. D., Chun, O. K., Kim Y. J., Moon, H. Y., Lee, different in vitro model systems. Food Chemistry, 115, 79—85.
Ch. Y., 2003: Quantification of phenolics and their antioxidant 12. Singleton, V. I., Ortofer, R., Lamuela-Raventos, R. M.,
capacity in fresh plums. J. Agric. Food Chem., 51, 6509—6515. 1999: Analysis of total phenols and other oxidation substances and
8. Kozuma, K., Tsuchiya, S., Kohori, J., Tokimitsu, I., antioxidants by means of Folin-Ciocalteu reagent. In Packer, L.
2005: Antihypertensive effect of green coffee bean extract (Ed.): Methods in Enzymology. Academic Press, Orlando, 152—178.
on mildly hypertensive subjects. Hypertension Research, 28,
711—718. ***
9. Krishnaiah, D., Sarbatly, R., Nithyananadam, R., 2011:
A review of the antioxidant potential of medicinal plant species. Selected paper from the 59th STUDENT SCIENTIFIC CON-
Food and Bioproducts Processing, 89, 217—233 FERENCE, Section III – Food hygiene and the environment, held at
10. Nikoli, M. C., Anese, M., Manzocco, L., Lerici, C. R., the University of Veterinary Medicine and Pharmacy in Košice, SR,
1997: Antioxidant properties of coffee brews in relation to the on April 6, 2016.
roasting degree. The LWT — Food Science and Technology, 30,
292—297.

38