Antioxidant and Antiradical Activity of Coffee

Abstract

This review summarizes published information concerning the determination of antioxidant activity (AA) in coffee samples by various methods (ORAC, FRAP, TRAP, TEAC, etc.) in vitro and limited data of antiradical activity of coffee products in vitro and in vivo. Comparison is carried out of the AA of coffee Arabica and coffee Robusta roasted at different temperatures as well as by different roasting methods (microwave, convection, etc.). Data on the antiradical activity of coffee is provided. The antioxidant activity of coffee, tea, cocoa, and red wine is compared. At the end of this review, the total antioxidant content (TAC) of coffee samples from 21 coffee-producing countries as measured by an amperometric method is provided. The TAC of green and roasted coffee beans is also compared.

Full text

Antioxidants 2013, 2, 230-245; doi:10.3390/antiox2040230
antioxidants
ISSN 2076-3921
www.mdpi.com/journal/antioxidants
Review
Antioxidant and Antiradical Activity of Coffee
Alexander Yashin
1
, Yakov Yashin
1
, Jing Yuan Wang
2
and Boris Nemzer
2,
*
1
InterLab, Inc., Selskohozyaistvennaya 12a, Moscow 129226, Russia;
E-Mails: yashinchrom@mail.ru (A.Y.); yashin@interlab.ru (Y.Y.)
2
VDF FutureCeuticals, Inc., 2692 N. State Rt. 1-17, Momence, IL 60954, USA;
E-Mail: rwang@futureceuticals.com
* Author to whom correspondence should be addressed; E-Mail: bnemzer@futureceuticals.com;
Tel.: +1-815-507-1400; Fax: +1-815-550-0013.
Received: 9 August 2013; in revised form: 27 September 2013 / Accepted: 29 September 2013 /
Published: 15 October 2013
Abstract: This review summarizes published information concerning the determination of
antioxidant activity (AA) in coffee samples by various methods (ORAC, FRAP, TRAP,
TEAC, etc.) in vitro and limited data of antiradical activity of coffee products in vitro and
in vivo. Comparison is carried out of the AA of coffee Arabica and coffee Robusta roasted
at different temperatures as well as by different roasting methods (microwave, convection,
etc.). Data on the antiradical activity of coffee is provided. The antioxidant activity of
coffee, tea, cocoa, and red wine is compared. At the end of this review, the total antioxidant
content (TAC) of coffee samples from 21 coffee-producing countries as measured by an
amperometric method is provided. The TAC of green and roasted coffee beans is also compared.
Keywords: antioxidants; antioxidant activity; ORAC; amperometry; coffee
1. Introduction
Studies carried out in recent decades have confirmed that excessive accumulation of oxygen and
nitrogen reaction products in body fluids including free radicals, such as the superoxide anion,
hydroxyl radical, hydroperoxyl radical, etc., is a major cause of pathological changes in the human
body, resulting in premature aging and numerous diseases.
The steady increase of free radicals in cells creates the conditions for so-called oxidative stress,
wherein free radicals oxidize blood vessel walls, protein molecules, DNA, carbohydrates, and lipids.
OPEN ACCESS

Antioxidants 2013, 2 231
These radicals are particularly active in interacting with membrane lipids that contain unsaturated
bonds, and thus alter the properties of cell membranes. The most active free radicals break bonds in
DNA molecules and damage the cells’ genetic apparatus regulating their growth, which can result in
cancerous cells. Oxidized low-density lipoproteins can be deposited on blood vessel walls which leads
to atherosclerosis and cardiovascular disease [1–3].
Oxidative stress also plays a key role in the pathogenesis of aging [4,5]. Oxidative stress can be
caused by various negative impacts, such as gamma or UV radiation, environmental factors, polluted
and poor-quality food, stress, some medications or treatments, smoking, alcoholism, etc.
Prolonged oxidative stress inevitably leads to dangerous diseases—such as cancer, cardiovascular
diseases, or diabetes—and premature aging. Oxidative stress can be reduced by antioxidant therapy,
i.e., by consumption of certain amounts of natural antioxidants contained in vegetables, fruits, berries,
vegetable oils, honey, tea, coffee, cocoa, juices, wine, sprouted grains, and other foods [6–8]. However,
in order to control consumption of antioxidants, it is necessary to know their content in foods and
beverages. In this regard, quantitative measurement of antioxidants in foods and beverages and
compilation of a corresponding databank becomes a highly important objective. Coffee is one of the
major sources of antioxidants in people’s daily diet.
2. Review of Publications on Determination of Coffee Antioxidant Activity in Vitro
Beneficial health effects of coffee are usually attributed to its high antioxidant activity (ability to
inhibit the process of oxidation). Many publications provide comparison of the antioxidant activity in
such popular beverages as coffee, tea, and cocoa [9–11].
Antioxidant activity of coffee is related to chlorogenic, ferulic, caffeic, and n-coumaric acids
contained in it [12]. In roasted coffee, melanoidins (brown pigments) are synthesized—these are strong
antioxidants [13]. In some publications, caffeine and trigonelline are considered to be antioxidants
also [14]. Phenylalanines which are formed during the roasting process show high antioxidant activity [14],
as do heterocyclic compounds [15].
Many publications explore the relationship between coffee antioxidant activity and roasting, for example:
–Comparison of antioxidant activity in green coffee beans with roasted coffee [16,17];
–Dependence of antioxidant activity on roasting temperature and time [17];
–Relation between the antioxidant activity and roasting method used (convection and microwave
roasting) [17].
Many researchers believe during the roasting process that Maillard reaction products, which are also
strong antioxidants, are formed [12,16,17].
The antioxidant activity of volatile compounds, such as furans, pyrroles, formed by pyrolysis of
coffee, has also been studied [14].
Interestingly, the compound, such as chlorogenic acid and polyphenols, which contributed to the
antioxidant activity in coffee, is geographically related [18]. The coffee fruit was found to have more
chlorogenic acids (CGA) in Arabica coffee fruit planted in Mexico and India compared to the coffee
fruit grew in China.

Antioxidants 2013, 2 232
In addition, evidence indicates that extraction procedures could affect the antioxidants contents in
coffee fruit as well as the caffeine content [19]. It has been shown that the antioxidant activity was
high in coffee fruit extract with low caffeine concentration in comparison with coffee fruit powder.
Apart from coffee, the antioxidant activity after roasting was also measured in wheat, nuts, and
some other food products.
The antioxidant activity of green and roasted coffee is compared in several publications [20–22].
3. Methods of Measuring the Antioxidant Activity and Antioxidant Content in Coffee
Many methods based on new reagents, model systems and devices were suggested to use for
determining the antioxidant activity, and many reviews were published on this subject [23–27].
The antioxidant activity (AA) is measured by various chemical and physicochemical methods.
All those methods are most often based on the direct or indirect measurement of reaction rate
and/or completeness.
Three types of methods could be distinguished based on the following measurements:
–Oxygen intake;
–Formation of oxidation products;
–Uptake or binding of free radicals.
In the first and second cases, AA is determined based on the degree of inhibition or intake rate for
reagents or the products formed.
Primary methods for the AA measurements are: ORAC—oxygen radical absorbance capacity;
TRAP—total radical trapping antioxidant parameter; FRAP—ferric reducing antioxidant power; TEAC
(Randox)—trolox equivalent antioxidant capacity; ABTS—2,2-azinobis(3-ethylbenzthiazoline)-6-
sulfonic acid; TBARS—thiobarbituric acid reactive substance.
In all of these methods, the AA depends on multiple parameters, including time, temperature, nature
of the substance, concentration of the antioxidants and other compounds, etc.
The antioxidant activity cannot be measured directly—what is typically measured is the effect of
the antioxidants on the degree of oxidation. All of these methods often give conflicting results.
The drawback of many methods used for measuring antioxidant activity is the lack of proper substrates
during the measurement process. Antioxidant activity is most often measured based on long-living
synthetic free radicals (ABTS, DPPH, AAPH, etc.). Lots of synonymous terms have been
proposed, including “antioxidant ability”, “antioxidant power”, “antioxidant activity”, and “antioxidant
capacity” [28]. All of these terms are related to the antioxidant concentration (the activity of substances or
substance groups).
Many well-known methods, such as TEAC, TRAP, FRAP, etc., are based on reduction reactions of
long-living free radicals or Fe (III) complex.
Obviously, all these methods have drawbacks because they use synthetic free radicals which have
nothing in common with free radicals in the human body.
The above methods are time-consuming and include several stages; the signals are recorded using
expensive devices, such as spectrophotometric, fluorimetric, and chemiluminescent detectors.

Antioxidants 2013, 2 233
Electrochemical methods are more promising for the measurement of integral antioxidant capacity
because the reaction between active oxygen compounds in aqueous media is accompanied by electron
transfer, i.e., they are electrochemical in nature.
In [29] has been proposed to measure antioxidant capacity by using electrogenerated oxidizers:
chlorine, bromine, and iodine. The electrogeneration of halogens during coulometric titration was performed
at a constant current of 5.0 mA from 0.2 M aqueous solutions of KCl and KBr in 0.1 M H
2
SO
4
; titration
end-point was detected by amperometric indication using two polarized platinum electrodes. The
authors of this method were the first to introduce a characteristic called “bromine-scavenging antioxidant
capacity” which is expressed in electricity units (coulombs) used for the titration of 100 g (or 100 mL)
of the tested material by electrogenerated bromine.
It was demonstrated that the value of bromine-scavenging antioxidant capacity reflects the total
content of antioxidants in foods, beverages, and extracts of medicinal plants. It should be noted, however,
that bromine and iodine can oxidize not only the antioxidants but also other compounds.
In one publication, it has been proposed to measure antioxidant activity by a potentiometric method
using a mediator solution of K
3
[Fe(CN)
6
]/K
4
[Fe(CN)
6
] [28].
This method was successfully used for the determination of the antioxidant activity of pure chemical
compounds as well foods, beverages, dietary supplements, and extracts of medicinal plants.
Another electrochemical method proposed is cathodic stripping voltammetry. In this method, oxygen
electroreduction is used as a model reaction.
In order to determine antioxidants in the tested substances, the dependences of the first oxygen
reduction peak current on their concentration in the solution and the duration of the process.
In this method, a mercury film electrode (banned in many countries) was used as an indicator.
Antioxidant activity of many foods and biological samples have been measured by this method.
3.1. ORAC (Oxygen Radical Absorbance Capacity) Method
The ORAC, one of the methods most often used for the determination of antioxidant activity,
especially in the U.S., was proposed by Cao G. [30] and later has been automated and validated by
Prior, R., Ou, B. and other four different assays [31–35].
This method is based on measuring changes in fluorescence intensity vs. reaction time. Quantitative
measurement of the antioxidant activity is carried out by determining the area between two curves
which represent reaction with and without the antioxidant.
In the early tests, B-phycoerythrin was used as the fluorescent probe, which tended to show slightly
lower results because this protein reacted with polyphenols. That is why it was later suggested to rather
use fluorescein, a more stable fluorescent compound.
In this method, 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH) is used as the source of
peroxyl radicals. In the ORAC method, trolox, a water-soluble analog of vitamin E, is used as the
standard. Fluorescence intensity is measured at 515 nm with the excitation wavelength of 485 nm.
Changes in the fluorescence intensity are registered every minute over 35 min, both with and
without an antioxidant. Therefore, the total measurement lasts for 70 min. The results obtained for
each sample are then calculated using calibration curves and presented in mmol/g of dry matter.

Antioxidants 2013, 2 234
The overall mechanism of the ORAC method is as follows: In the solution, the AAPH reagent
decomposes at 37 °C with the release of peroxide free radical. When an antioxidant, or a mixture of
antioxidants, are added, fluorescence quenching decreases due to the fact that antioxidants neutralize
the effects of free radicals on the fluorescein. The convergence of the method is in the form of standard
deviation (SD) ± 15%.
In a 2005, Prior, et al. [36] wrote that a standardized total antioxidant capacity method should
evaluate “effectiveness against various ROS/RNS such as superoxide anion, hydroxyl, and
peroxynitrite...and this may require additional methods specific for each radical source”.
The new generation of ORAC assay–total ORAC for food and nutrition (Total ORAC FN) has been
introduced by Brunswick Laboratories (Norton, MN, USA) in 2008. Total ORAC FN represents a
breakthrough in comprehensive antioxidant testing for food and nutrition products as antioxidant
activity against five of the most important free radicals found in human–hydroxyl (HORAC assay),
peroxyl (ORAC assay), peroxynitrite (NORAC assay), singlet oxygen (SOAC assay), and superoxide
anion (SORAC assay).
The total antioxidant capacity of whole coffee fruits Arabica and their extracts commercially produced
by FutureCeuticals, Inc. (Momence, IL, USA) has been analyzed using Total ORAC FN assays [19].
The results of ORAC FN are shown in Table 1.
Table 1. Total ORAC activity coffee fruit sample [19]. (Note: ORAC FN results are
expressed as µmole Trolox Equivalent (TE)/g ± SD.
Coffee Fruit Sample Coffee Fruits Extract-1
Coffee Fruits
Extract-2
Coffee Fruits
Powder-1
Coffee Fruits
Powder-2
Extration/Drying
Procedure
Multistep ethanol
extraction
Single step extraction Freeze-dried Air-dried
Total CGA 76.5% 45.0% 8.79% 4.53%
ORAC 15,246 ± 453 6097 ± 225 823 ± 86 735 ± 47
HORAC 41,389 ± 3447 18,709 ± 426 3520 ± 287 2140 ± 125
NORAC 1317 ± 104 527 ± 52 75 ± 11 52 ± 7
SORAC 2193 ± 1591 860 ± 24 271 ± 14 123 ± 62
SOAC 3422 ± 355 2042 ± 185 311 ± 13 239 ± 12
Total ORAC FN 64,354 ± 2584 28,237 ± 782 4768 ± 285 3439 ± 134
Table 1 shows that the total chlorogenic acid (CGA) contents correlates to total antioxidant
capacity. The coffee extract with the highest chlorogenic acid contents corresponds to the highest total
ORAC activity compared with whole coffee fruit powder (air dried and freeze dried) [19].
3.2. Amperometric Method for Determining Total Antioxidant Content
Amperometric method (AM) is based on measuring an electric current in the detector cell which
occurs during oxidation of the analyte on the working electrode surface when certain potentials are
applied. Thus, when an amperometric method is used, the changes in the current passing through the
cell are registered—these changes directly correlate to the changes in the analyte concentration [1].
The amperometric method can operate in three modes: at a constant potential, at pulse potentials,
and by scanning the potentials in the entire range.

Antioxidants 2013, 2 235
The amount of electric current depends on the nature of the analyte, nature of the working electrode,
and the potential applied to the electrode.
Emerging electric currents are very small, within 10
−6
to 10
−10
A. These analog signals are amplified,
converted into digital signal by an analog-digital converter (ADC), and then displayed on a computer
screen. If necessary, the outputs can be printed.
The working electrode is a glassy carbon electrode which is most commonly used in the determination
of polyphenolic compounds. Ionization potentials of phenolic compounds vary within 100–1300 mV.
The signal is recorded as differential output curves. Using special software, the areas or peak heights
(of the differential curves) are calculated for the analyte and for the reference substance. Well-known
antioxidants, such as quercetin, dihydroquercetin, mexidol, trolox, gallic acid, etc. can be used as
reference substances. The amperometric method has several advantages in the determination of antioxidant
activity: not taking into account sample preparation, one determination takes only a few min; analysis
(data recording and processing) takes place in real time; accuracy and reproducibility of the analysis is
ensured by accurate dosing with a six-way valve; standard deviation (SD) of valve dispenses is less
than 0.5%; SD of the successive measurements of the analyzed samples is less than 5%; limit of
detection for polyphenols and flavonoids is at the level of nanograms and picograms (10
−9
–10
−12
g). At
such low concentrations, the likelihood of the mutual influence of different jointly present antioxidants,
such as by a manifestation of synergy, is significantly reduced.
At the same time, high selectivity in determination only of antioxidants (compounds capable of
inhibiting oxidation) is achieved; other compounds present in complex mixtures do not interfere with
the determination of the antioxidants. No chemicals (other than standards) are required for this
analysis; therefore, the cost of measurements is very low.
The amperometric method is the only method which allows for direct measurement of all antioxidants
in a sample. The amperometric method has been successfully applied to determine the antioxidant
capacity of various wines and coffee [37]. It was noted that this method is direct, accurate, objective,
and fast. In one publication, this method was used to determine the antioxidant capacity of olive oil,
i.e., fat-soluble samples, from different Mediterranean countries—Italy, Greece, France, Spain, Morocco,
Tunisia [38]. This method allows for the assessment of quality and authenticity of olive oil. In one study,
the antioxidant power of lipophilic compounds present in vegetables, such as carotenoids, chlorophyll,
tocopherols and capsaicin, was determined [39]. When applied to pure compounds, the order of antioxidant
power resulted as follows (in descending order): lycopene > beta-carotene > zeaxanthin > alpha-carotene >
beta-cryptoxanthin > lutein > α-tocopherol > capsaicin > chlorophyll a > chlorophyll b > astaxanthin >
canthaxanthin. The results of the antioxidant activity measurements obtained for five vegetable and
two fruit extracts were compared to those obtained by the ABTS. A good correlation between the two
methods was found, except for spinach. The authors conclude that the amperometric method can be
successfully used for direct, quick, and reliable monitoring of antioxidant power of lipophilic food extracts.
Amperometric (electrochemical) method is also used to determine the antioxidant status in people.

Antioxidants 2013, 2 236
4. Results of Antioxidant and Antiradical Activity of Coffee
4.1. Antioxidants and Antioxidant Activity
The most antioxidant-rich beverages are [1]: coffee—200–550 mg/cup; tea—150–400 mg/cup;
red wine—150–400 mg/glass. Intake of these drinks makes a significant contribution to the total
amount of antioxidants consumed by people.
Green coffee beans contain large amounts of polyphenolic antioxidants, such as chlorogenic,
caffeic, ferulic, and n-coumarinic acids. Coffee roasting significantly alters the composition of polyphenols
due to Maillard reaction (chemical reaction between amino acids and sugars).
A cup of coffee which contains 10 g of roasted coffee beans may have 15 to 325 mg of chlorogenic
acids. On average in America, one cup contains approximately 200 mg of chlorogenic acid. Antioxidant
activity of ferulic and caffeic acids was studied both in vitro and in vivo. Ferulic acid presented in
coffee has anti-inflammatory, anti-allergic, antibacterial, antiplatelet, and antiviral effect [34].
Pharmacological properties of ferulic acid are related to its high antioxidant activity, in particular, its
ability to inhibit lipid peroxidation in biological membranes.
In one study, it has been shown that ferulic acid at a concentration of 10
−3
mol/L in a perfusion
solution reduces arrhythmia [40].
People consume coffee or tea every day, sometimes several times a day. Coffee is the main drink in
Europe, America and Asia. In recent years, consumption of coffee has increased even in England, a
prominently tea-drinking country. Coffee consumption is rising rapidly in China and Japan where a
centuries-old tradition of tea consumption is strongest.
Coffee and tea provide a very significant portion of the daily intake of antioxidants established
for humans.
We have compared coffee, tea and cocoa in terms of the total antioxidant content (TAC). The
results of the comparison are shown in the Table 2.
Table 2. Total antioxidant content of different beverages. Note: Natural roasted coffee
produced by the Madeo Company. Best grades of green and black tea purchased in the
stores. (Averaged values of TAC) [41].
Beverages One-time consumption rate, in grams TAC, mg/g
Coffee 7–10 150–300
Green Tea 2 150–300
Black Tea 2 110–200
Cocoa 10 200–250
The data presented in Table 2 suggest that in terms of antioxidant capacity (as total antioxidant
content), coffee and cocoa are comparable with tea, which can be attributed to higher consumption
rates of these products. The antioxidant capacity of tea is largely related to the catechins and their
polymers (thearubigins, etc.).
Cocoa is the most balanced product in terms of antioxidants since it contains water-soluble and
lipid-soluble antioxidants. This point towards an interesting combination of coffee and cocoa and some
manufactures are attempting to create such mixtures.

Antioxidants 2013, 2 237
Table 3 summarizes data obtained in Italy on the antioxidant activity of various non-alcoholic
and alcoholic beverages [42]. It should be noted that figures provided for green and black tea as well
as for beer are understated. According to our data, the antioxidant activity of cognac is higher than that
of whisky.
Table 3. Comparison between the antioxidant activity (AA) of coffee and other
drinks [42] (Notes: All samples were collected in Italy; FRAP—ferric reducing antioxidant
power; TRAP—total radical-trapping antioxidant parameter; TEAC—trolox equivalent
antioxidant capacity).
No. Beverages
AA determined by different methods
FRAP (mol Fe
2+
/L) TRAP (mol Trolox/L) TEAC (mol Trolox/L)
1. Coffee (Espresso) 129.4 66 36.5
2. Coffee (Instant) 108.6 52.4 32.5
3. Coffee (Extract) 96.4 59.6 30.3
4. Coffee (Espresso, Decaffeinated) 93.0 45.8 27.0
5. Red wine (Chianti) 31.5 14.8 11.4
6. Green Tea 18.0 7.6 6.0
7. Black Tea 10.1 4.9 3.6
8. Rose wine (Villa Tofre) 8.3 2.2 2.4
9. White wine (Pinot) 3.7 2.1 1.7
10. Whisky 3.4 2.3 1.7
11. Cognac 2.2 1.5 1.3
12. Beer 2.8 – 1.0
Data of caffeine, theobromine, and total polyphenols content in standard cups of coffee and tea, as
well as their antioxidant activity, are provided in Table 4 [20].
Table 4. Caffeine, theobromine and total polyphenols content in one cup of coffee and
tea [20]. (Notes: Caffeine and theobromine were determined using high performance
liquid chromatography (HPLC); the total content of polyphenols was determined by
Folin-Ciocalteu assay).
No. Description Coffee Tea
1. Caffeine, mg/cup 181 ± 10 130 ± 7
2. Theobromine, mg/cup 28.9 ± 1.1 5.9 ± 0.4
3. The total content of polyphenols, gallic acid equivalents (GAE) mg/cup 161 ± 9 87 ± 9
4. AA, Crocin test, mM TX equivalent/cup 7.4 ± 0.3 1.4 ± 0.1
5. TRAP, mm ROO° equivalent/cup 10.1 ± 0.6 1.3 ± 0.1
Table 5 shows the effect of the roasting degree of Arabica and Robusta coffee beans on their
antioxidant activity [11]. The antioxidant activity of the beverage is represented by the increase of the
lag time (lag time of LDL in the presence of coffee beverage with respect to the lag time of control
LDL). Results are expressed as mean (of three to five individual experiments) ± SD.

Antioxidants 2013, 2 238
Table 5. Antioxidant activity of green and roasted Arabica and Robusta coffee beans [11].
No. Type of Coffee
Increase of lag time, min
Arabica Robusta
1. Green 366 ± 74 643 ± 68
Roasting degree:
2. −light 284 ± 80 294 ± 41
3. −medium 206 ± 30 190 ± 39
4. −dark 168 ± 23 134 ± 34
As can be seen from Table 5, the antioxidant activity of Robusta green coffee is significantly higher
than that of Arabica. However, this difference virtually disappears after light roasting; and after dark
roasting Arabica coffee even exceeds Robusta coffee with regards to antioxidant activity.
In one article, antioxidant activity of coffee, tea, and cocoa—the most widely consumed beverages
containing polyphenolic antioxidants–was compared [11]. The beverages were prepared as 0.7%–2.5%
soluble coffee and 1.5%–3.5% cocoa; teas (green, black, or herbal) were prepared as one tea bag
brewed in 220 mL of hot water. These drinks are often taken with milk. It has been shown that addition
of milk did not alter the antioxidant activity.
The currently used roasting methods—convection and microwave roasting—were compared in [17].
Data on the antioxidant activity measured by spectrophotometric (UV-vis) and HPLC methods, as
well as data on caffeine content in various types of coffee and in coffee roasted by different methods,
is presented in Table 6.
Table 6. Total polyphenols and caffeine content of raw and different roasted coffee beans [17].
No. Type of coffee and roasting method
Total amount of polyphenols, %
Caffeine, %
UV HPLC
1. Green 7.88 7.15 2.12
Roasted:
2. −convection 2.26 2.07 1.82
3. −microwave 2.31 2.19 1.90
4. −wet, convection 2.58 2.36 1.89
5. −wet, microwave 2.75 2.64 2.07
6. −dry, convection 2.06 1.97 1.86
7. −dry, microwave 2.11 2.03 2.08
8. −convectio microwave 3.03 2.87 1.95
As can be seen from Table 6, microwave roasting preserves antioxidants better than convection
roasting, and the wet method for separating the beans from the fruits is preferable to the dry method by
this criterion. The latter is probably related to the greater oxidation of the antioxidants in the dry method.
In one study, efficiency of extraction of biologically active compounds from the coffee powder was
researched [43]. Extraction was carried out with ethyl acetate, ethyl ether, and dichloromethane. Ethyl
acetate in acidic medium was shown to be the most effective.
The yellow-brown extract of Robusta coffee was separated by gel-filtration chromatography according
to the molecular weight. Individual fractions were collected at the column outlet for further study of

Antioxidants 2013, 2 239
their antioxidant activity (using a β-carotene-lipoic acid model system). Protective activity (PA)
against lipid oxidation was determined in vitro. The results of these studies are summarized in Table 6.
As can be seen from Table 7, only high-molecular weight fractions showed antioxidant activity and
only low-molecular weight fractions had protective activity. These interesting data require further
careful research.
Table 7. Dry residue, molecular masses, antioxidant activity (AA%), and protective activity
(PA%) of coffee fractions obtained by gel filtration chromatography (GFC) of acidic
extract [43].
No. Sample mg/mL
a
Molecular weight AA, % PA, %
1. acidic coffee extract 1.31 – 96 100
2. 1st fraction 0.50 <6000 74 0
3. 2nd fraction 0.16 <550 37 0
4. 3rd fraction 0.13 350–550 0 0
5. 4th fraction 0.23 200–350 0 53
6. 5th fraction 0.29 <200 0 85
Note:
a
mg of dry residue per mL of brewed coffee.
In that same study, the pH of the coffee solutions (green and varying degrees of roasting) was also
determined. Robusta coffee varieties from Zaire and Ecuador and Arabica coffee varieties from Brazil,
Colombia, and Costa Rica were studied. According to the results of this research, all varieties of green
coffee have a higher pH than roasted coffees. Values ranges from 5.77 to 5.95. For roasted Arabica
coffee varieties the pH values were lower than for Robusta varieties: 5.01–5.39 and 5.43–5.68, respectively.
As for the antioxidant activity, it is higher in green coffee than in roasted; at the same time, for all
coffee varieties its value increased by 4%–6% with the increase of the roasting time (from 10 to 30 min).
4.2. Antiradical Activity
It should be emphasized that there is a difference between “antiradical” and “antioxidant” activity.
The antiradical activity characterizes the ability of components to react with free radicals (in a single
free radicals reaction), but antioxidant activity represents the ability to inhibit the process of oxidation
(which usually involves a set of different reactions) [44].
In [45] have been shown that solutions of green and roasted coffee have significant antiradical
activity against hydroxyl free radicals. This effect was studied both in vitro (using deoxyribose assay)
and in vivo in biological cellular systems (IMR32 cells).
All solutions tested in vitro using the deoxyribose degradation assay have shown similar inhibitory
activity. In the cell cultures, solutions of Robusta roasted coffee have shown greater activity than other
solutions. Using preparative gel-filtration chromatography, the components of the roasted Robusta
coffee were separated into three fractions by their molecular weight. The most active fraction was
found to be the one containing the 5-O-caffeoylquinic acid. Activity of the solution increased when
concentration of this acid increased (from 0.02 to 0.10 mM).
When roasted, the content of chlorogenic acids decreases but at the same time, the content of high
molecular compounds, and melanoidins (also possessing antiradical activity in coffee) increases.

Antioxidants 2013, 2 240
In order to confirm that the 5-O-caffeoylquinic acid has the greatest efficiency, solutions of coffee
biologically active compounds were prepared and tested for their antiradical activity (see Table 8).
Table 8. Antiradical activity in vitro and in vivo of standard solutions of coffee
biologically active components [45].
No. Compounds
%
in vitro in vivo
1. 5-O-caffeoylquinic acid 34.6 ± 7.5 264.3 ± 56.2
2. Caffeine 27.2 ± 6.4 25.2 ± 5.2
3. Nicotinic acid 19.2 ± 6.7 23.2 ± 4.8
4. Trigonelline hydrochloride 19.3 ± 12.6 15.7 ± 4.1
Table 8 shows that 5-O-caffeoylquinic acid has the highest activity both in vitro and in vivo,
particularly, in vivo. These results could explain the significant neuroprotective effects found for coffee
consumption in recent epidemiological studies. These studies have established a direct link between
coffee consumption and reduced risk of neurodegenerative diseases, including Parkinson’s disease [46–48].
Coffee and coffee beverages can inhibit in vitro the mutagenicity of oxidants, such as tert-butyl
hydroperoxide, and therefore can inhibit lipid peroxidation and formation of malondialdehyde, a marker
of oxidative stress [49].
4.3. Total Antioxidant Content by Amperometric Method
The amperometric measurements of total antioxidant content has been provided by amperometric
detector or analyzer TsvetYauza-01-AA (Khimavtomatika, Moscow, Russia).
The device operates on specially selected modes whereby only antioxidants are determined, and
other compounds do not interfere with their determination.
Samples to be used for the determination of the total content of antioxidants in coffee were prepared
as follows: the roasted coffee beans were ground into a powder, then boiling water was added to
the powder, and the mixture was brewed for 5–10 min. A sample was taken using a syringe and
introduced to a six-way valve on the device. When the valve was turned from the “dosing” position to
the “analysis” position, the mobile phase (eluent) captured the sample and transferred it to the cell of
the amperometric detector.
The total antioxidant content (TAC) in roasted coffee beans (provided by coffee manufacturer
Freshly Roasted Company; coffee varieties of the MADEO trademark) from major coffee producers in
21 countries, is shown in Table 9. Quercetin was used as a reference substance.
As can be seen from Table 9, the content of antioxidants in different varieties of coffee differs 1.5 times
at most and depends on the country of origin, degree of roasting, coffee processing method, altitude at
which the coffee was grown, the soil, the average temperature, and number of sunny days per year.
Drinking two or three cups of coffee made with roasted beans ensures the daily consumption rate
for antioxidants. Unfortunately, only 20% of consumers use natural roasted coffee in Russia, whereas,
for example, in the U.S., this figure is 80%. In recent decades, instant coffee grades have become
widely used. True lovers of coffee, “coffee nerds”, undoubtedly prefer natural roasted coffee because
this type of coffee has a unique taste and aroma.

Antioxidants 2013, 2 241
Table 9. The total content of antioxidants (TAC) in MADEO coffee beans provided by
company “Freshly Roasted Coffee” (quercetin used as standard) [41,50].
No. Name TAC, mg/g TAC, mg/7 g
(per cup)
1 Maragogype, Guatemala 32.1 224.7
2 Brazil 30.8 215.6
3 Colombia 30.5 213.5
4 Yirgacheffe, Ethiopia 28.6 200.2
5 Puerto Rico 28.6 200.2
6 Decaffeinated coffee 27.3 191.1
7 Kenya 27.0 189.0
8 Cuba 26.7 186.9
9 Jamaica 26.1 182.7
10 Sumatra 24.5 171.5
11 Mexico 24.1 168.7
12 Nicaragua 24.1 168.7
13 Guatemala 24.0 168.0
14 Honduras 23.9 167.3
15 Maragogype, Nicaragua 23.2 162.4
16 Maragogype, Mexico 23.1 161.7
17 Tanzania 23.1 161.7
18 Yemen 22.9 160.3
19 Costa Rica 22.5 157.5
20 Skybury, Australia 22.3 156.1
21 Java 21.1 147.7
Due to some concentration during the extraction procedure, some varieties of instant coffee may
contain greater amounts of antioxidants than roasted coffee.
The TAC values for green and roasted coffee beans from several different countries in Africa, South
America, and Asia are provided in Table 10. As can be seen from Table 9, Arabica roasted coffee
beans showed higher TAC than green coffee beans, which is quite an unexpected result. Previously,
similar results were observed in some studies which used other methods.
Table 10. Impact of coffee roasting process on the total antioxidant content (quercetin used
as standard) [41,50].
No. Type of Coffee Producing Country
TAC, mg/g
Green Roasted
1. Arabica Tanzania 34 43
2. Arabica Ethiopia (Yirgacheffe) 32 40
3. Arabica Brazil 30 41
4. Robusta Bali 65 63
5. Robusta Uganda 67 55

Antioxidants 2013, 2 242
5. Conclusions
In the publications reviewed, the values for both antioxidant activity (AA) and total antioxidant
content (TAC) of roasted coffee are at the same level as corresponding values for tea, cocoa, and red wine.
Roasting has a contradictory effect on TAC and AA. The HPLC results show that the content of
chlorogenic acids, the main antioxidants in green coffee beans, significantly decreases after roasting.
On the other hand, in the process of roasting such polymeric compounds as melanoidins (which are
potent antioxidants) and other compounds are formed.
Conflicts of Interest
The authors declare no conflict of interest.
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