Journal of the Science of Food and Agriculture
J Sci Food Agric 87:1069–1075 (2007)
Antioxidant capacity of Spanish honeys
and its correlation with polyphenol
content and other physicochemical properties
Lucı́a Vela, Cristina de Lorenzo and Rosa Ana Pérez∗
Departamento de Investigación Agroalimentaria, Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA),
Finca El Encin, Aptdo 127, E-28800 Alcalá de Henares, Madrid, Spain
Abstract: Thirty-six Spanish honeys of different floral origins (nectars and honeydews) were assessed to
estimate their radical-scavenging capacity against the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl),
their ability to inhibit enzymatic browning in apple homogenate and their clarifying effect on apple juice.
These capacities were evaluated spectrophotometrically. Physicochemical parameters such as pH, acidity, net
absorbance, electrical conductivity and total polyphenol content of the samples were also evaluated. It was observed
that all these parameters presented a strong correlation with radical-scavenging capacity, whereas antibrowning
capacity had a low correlation with conductivity and no correlation with honey pH. When data of nectar and
honeydew honeys were evaluated separately, net absorbance and honey acidity seemed to be good parameters to
evaluate the antioxidant activity of nectar honeys; however, in honeydew honeys there were only relationships
between radical-scavenging activity and honey conductivity and between browning inhibition of homogenates and
total acidity. In general, honeydew honeys showed higher antioxidant capacities than nectar honeys.
 2007 Society of Chemical Industry
Keywords: honey; antioxidant activity; DPPH; enzymatic browning
INTRODUCTION
Oxidative stress, which leads to the oxidation of
low-density lipoproteins, occurs when free radicals
and reactive oxygen species in a system are not
in balance and their production exceeds the ability
of the system to neutralise and eliminate them.
There is increasing evidence that free radicals
contribute to the development of diseases such
as neurodegenerative disease, chronic inflammatory
disease, cancer, cardiovascular disease and aging.1
Antioxidants are molecules or compounds that act
as free radical scavengers. For this reason, in recent
years there has been an increasing demand for
natural antioxidants as food preservatives and for
the prevention of several diseases. Thus antioxidants
in vegetables,2 – 4 fruits,5,6 drinks,7,8 olive oil9 and
grains10,11 have attracted much attention. In addition,
although scarce in the literature, some antioxidant
activity studies on honeys from different countries
and of different botanical origins have been carried
out.12 – 15
Numerous in vitro methods are used to
evaluate the antioxidant potential of natural
products. For this reason, some studies have
been carried out using more than one of
the following techniques in order to compare
results: ABTS (2,2 -azino-bis(3-ethylbenzothiazoline6-sulfonic acid) assay,8,16,17 DPPH (1,1-diphenyl2-picrylhydrazyl radical) test,17,19,20 ORAC (oxygen
radical absorbance capacity) test17,19,21 and FRAP
(ferric-reducing antioxidant power) assay.16,22 Most
of them are based on the presence of free radicals in
the reaction mixture and measurement of the relative
effects of added antioxidant on some properties of
the mixture, comparing these effects with the capacity
shown by a standard mixture.
Honey is a complex natural product produced
by honeybees from the nectar of blossoms or from
exudates of trees and plants, usually with the
participation of plant-sucking insects. These different
botanical origins give rise to nectar or honeydew
honeys respectively. Honey has been used since
ancient times mainly as a sweetening agent, but it
has also been employed in a therapeutic capacity. The
high sweetening power of honey is due to the presence
of the monosaccharides fructose and glucose as main
components (60–85%); however, the components
involved in its therapeutic properties have not yet been
determined. Thus honey is a very complex product;
its composition is closely associated with its botanical
origin, with phenolic compounds, minerals, proteins,
∗
Correspondence to: Rosa Ana Pérez, Departamento de Investigación Agroalimentaria, Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y
Alimentario (IMIDRA), Finca El Encin, Aptdo 127, E-28800 Alcalá de Henares, Madrid, Spain
E-mail: [email protected]
Contract/grant sponsor: INIA (MCYT, Spain); contract/grant number: CAL02-012
Contract/grant sponsor: IMIDRA (Comunidad de Madrid)
Contract/grant sponsor: Spanish Ministerio de Ciencia y Tecnologı́a
(Received 26 April 2006; revised version received 20 July 2006; accepted 2 August 2006)
Published online 26 February 2007; DOI: 10.1002/jsfa.2813
 2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00
L Vela, C de Lorenzo, RA Pérez
free amino acids, enzymes and vitamins as minor
components.23
Honeys show a wide range of antioxidant activity
depending on their botanical origin; high correlations
have been described between this activity and honey
colour24 or polyphenolic content.12,14 The main
techniques used for evaluating the in vitro antioxidant
activity of honeys are DPPH12,15,25,26 and ORAC13,27
assays. Use of the ORAC methodology has increased
in recent years, because it minimises potential crossreactions between samples and reagents; however,
it requires a spectrofluorometric analyser. Thus,
although less selective, the DPPH assay is more widely
accepted, because it is a simple method that allows
evaluation of the radical-scavenging activity by means
of a spectrophotometer, which is common laboratory
equipment.
Enzymatic browning in foods, mainly associated
with polyphenol oxidase (PPO) activity, is the major
impact factor on food quality. Nowadays there is
a demand for minimally processed food without
synthetic chemical preservatives to prevent this effect.
Therefore the use of honey as a natural browning
inhibitor seems to be of great interest for the consumer.
Although studies on the antibrowning effect of honey
are scarce in the scientific literature,15,28 – 30 they have
shown honey as a potential natural antioxidant in
fruits. However, these assays were carried out with
only a few honey varieties, and it is known that the
antioxidant activity of honeys shows a wide range
of values, highly dependent on their botanical and
geographical origins.
The main aims of this work were (1) to evaluate the
antioxidant capacity of Spanish nectar and honeydew
honeys by analysing their radical-scavenging activity
and browning inhibition capacity and (2) to establish a
possible relationship between the antioxidant activity
of honeys and their physicochemical characteristics.
MATERIALS AND METHODS
Chemicals and honey samples
Folin–Ciocalteu reagent was obtained from Merck
(Darmstadt, Germany). Sodium hydroxide and
hydrochloric acid (35%) were purchased from Panreac (Barcelona, Spain). Gallic acid was obtained from
Carlo-Erba (Rodano, Itlay). Ascorbic acid and DPPH
were purchased from Sigma-Aldrich (St. Louis, USA).
A total of 36 samples of handicraft honeys from
different locations in Spain were collected directly
from beekeepers or from apiculture trade exhibitions
(Table 1).
Characterisation of the honeys was done through
melissopalynological and physicochemical analyses.
Melissopalynological analysis
Microscopic analysis of honey sediment composition
was performed essentially according to the method
described by Louveaux et al.31 using a non-acetolytic
technique, in order to preserve honeydew elements,
1070
Table 1. Geographical origins and numbers of different honey
samples studied
Region of
Spain
Number of
samples
Castilla-León
Extremadura
Galicia
2
3
6
Castilla-la Mancha
9
Aragón
Madrid
2
14
Type
Honeydew
Honeydew (2), rosemary (1)
Honeydew (3), heather (1),
eucalyptus (2)
Honeydew (2), rosemary (1),
lavender (3), multifloral (3)
Honeydew, lavender
Honeydew (7), echium (5),
heather (2)
with the modifications proposed by Terradillos et al.32
Microscopic observations were made using a Leica
DMR light microscope (Cambridge, UK) fitted with
a digital camera and coupled to an image analysis
system (Leica QWin Std software) for morphometric
measurements of pollen grains. Identification of pollen
grains was made using a collection of reference
pollens.33
Physicochemical analysis
Physicochemical parameters were determined according to the Harmonized Methods of the International
Honey Commission (IHC) as transcribed in the Spanish Official Methods for Honey Analysis.34 The parameters pH, electrical conductivity, ash content and
glucose plus fructose content were chosen, together
with the melissopalynological results, for an initial
classification of samples into nectar and honeydew
honeys. pH and acidities (free, lactonic and total) were
determined in solutions of honey (10 g) in deionised
water (75 mL) by endpoint titration. Moisture content was determined by refractive index measurement
at 20 ◦ C in an Abbe refractometer and correlation
with defined Chataway charts. Electrical conductivity
was measured with a Crison GLP31 conductimeter
(Barcelona, Spain) in a solution of 0.2 g mL−1 honey
dry matter in deionised water. Ash content was determined by calcination at 550 ◦ C until constant weight,
with the precaution of including a previous caramelisation step on a heating plate to control the production
of foams and sample losses. Net absorbance, as colour
parameter, was measured by UV–visible spectrophotometry (Perkin Elmer Lambda 10 spectrophotometer, Norwalk CT, USA) and defined as the difference between absorbance measurements at 560 and
720 nm.35 Glucose and fructose contents were determined by high-performance liquid chromatography
(HPLC) using a RECEX monosaccharide precolumn
and column (Phenomenex, Torrance, USA) at 90 ◦ C
and a refractive index detector. Elution was performed
using HPLC-grade water as eluent at a flow rate of
1 mL min−1 . Total polyphenol content was determined with Folin–Ciocalteu reagent by absorbance
measurement at 670 nm using gallic acid as standard.
J Sci Food Agric 87:1069–1075 (2007)
DOI: 10.1002/jsfa
Antioxidant capacity of Spanish honeys
DPPH radical-scavenging activity
The antioxidant activity of honey samples in the
presence of the stable free radical DPPH was
determined spectrophotometrically. Briefly, 1.25 mL
of honey solution in deionised water (0.025 g mL−1 )
was mixed with 1.5 mL of a 90 µg mL−1 solution
of DPPH in methanol. After a 5 min incubation
period the absorbance was read at 517 nm against
a water/methanol (1:1 v/v) blank. The scavenging
capacity of each honey was estimated using a standard
curve of ascorbic acid and the result given as %
equivalent of ascorbic acid, in terms of DPPH
depletion, which was calculated using the equation
scavenging activity (%)
= [Aa − (Ab − Ac )] × 100/(Aa − Ao )
where Aa is the absorbance obtained without the
honey sample (DPPH and methanol only), Ab is the
absorbance of the incubation mixture of DPPH and
honey solution, Ac is the absorbance of the blank
solution and Ao is the minimum absorbance obtained,
when DPPH was completely scavenged.
Effect of honey on minced apple browning
Fresh homogenates of Golden Delicious apples (10 g)
were assayed with five honey concentrations from 0.5
to 4% honey per homogenate (w/w). Two controls
were needed to determine the highest and lowest
browning: the homogenate without honey (highest
browning effect) and the homogenate after thermal
treatment in a boiling water bath, followed by a
refrigeration step, in order to inactivate PPO (lowest
browning effect). Samples were incubated for 1 h prior
to the addition of 20 mL of aqueous methanol (1:1)
and then mixed, filtered and centrifuged at 18500 × g
for 10 min. The absorbance of the extract was
determined at 420 nm against an aqueous methanol
blank.
Data are expressed as browning inhibition (%) of
each honey solution. Each result is the mean of three
replications.
Effect of honey on apple juice
Apple juice was prepared from Golden Delicious
using a conventional blender (Moulinex). The fresh
juice was immediately filtered under vacuum and the
filtered fraction placed in an ice bath to minimise juice
browning before the assay. Apple juice was added to
a vial containing honey to give a final concentration
of 4% honey. After 4 h of incubation the effect of
the honey was evaluated spectrophotometrically at
420 nm or by measurement of its CIE L∗ , a∗ , b∗
colour parameters. Spectrophotometric results were
expressed as % equivalent of ascorbic acid using two
reference values: the absorbance after incubation of a
mixture of juice and ascorbic acid (1%), i.e. the highest
juice clarification value (100% inhibition), and the
absorbance of the juice without the addition of honey,
J Sci Food Agric 87:1069–1075 (2007)
DOI: 10.1002/jsfa
i.e. the lowest juice clarification value. Interference of
the colour of honeys was avoided using honey blanks.
Each result is the mean of three replications.
In the colorimetric method, CIE L∗ , a∗ , b∗
colour parameters were measured with a Minolta
CR200 colorimeter (Minolta, Osaka, Japan). With
this method the colour differences detected were
calculated using the Euclidean distance between the
colour parameters with and without antioxidant. This
Euclidean distance can be computed as
E = [(La − Lb )2 (aa − ab )2 (ba − bb )2 ]1/2
where La , aa and ba are the values obtained for the
antioxidant assayed and Lb , ab and bb are the values
obtained without antioxidant (blank sample).36
Statistical analysis
Data were processed using SPSS for Windows
v12.0 (SPSS Inc., Chicago, Illinois). Unless specified
otherwise, a significance level of P < 0.05 was chosen
in the statistical analysis.
RESULTS AND DISCUSSION
The physicochemical characteristics of 36 Spanish
honeys of different botanical and geographical origins
were evaluated. The relation between some of these
parameters and the honey source has been considered
previously by other authors.37 – 39 The mean values and
ranges obtained for each physicochemical parameter
are summarised in Table 2. To compare the values
obtained for the two honey groups, a t test
for comparison of two means was carried out.
As expected, the majority of the physicochemical
characteristics evaluated showed high discrimination
between nectar and honeydew honeys: pH, acidity,
electrical conductivity, net absorbance, ash content
and polyphenol content (Table 2). Honeydew honeys
are characterised by higher values of these parameters
than nectar honeys. Although glucose plus fructose
content has been previously described as a highly
discriminatory variable,37 the results obtained here
did not demonstrate the capacity of this parameter to
distinguish between the two honey groups.
DPPH radical-scavenging activity
In this study the DPPH radical-scavenging capacity
of honeys was measured using ascorbic acid as
reference. The antioxidant activity of honey samples
was calculated as % DPPH decoloration.15,20 In
these analyses the addition of xylene described by
Chen et al.15 was evaluated. Xylene was added to
a mixture of honey and methanolic DPPH solution
previously incubated for 5 min; this was followed
by separation and centrifugation of the xylene layer.
The scavenging activity analyses carried out with and
without xylene addition yielded similar results (data
not shown). Thus scavenging activities were eventually
established by absorbance measurements exactly 5 min
1071
L Vela, C de Lorenzo, RA Pérez
Table 2. Average values and ranges of data obtained in physicochemical analysis of 36 honeys
Honeydew honeys
pH
Free acidity (meq kg−1 )
Lactonic acidity (meq kg−1 )
Total acidity (meq kg−1 )
Electrical conductivity (10−4 S cm−1 )
Ash (%)
Fructose + glucose (%)
Polyphenol content (mg kg−1 honey)
Net absorbance (A560 − A720 )
a
Nectar honeys
Mean
Maximum
Minimum
Mean
Maximum
Minimum
4.64
43.2
4.92
48.1
10.06
0.59
64.68
1.13
0.68
5.08
58.4
27.66
64.3
12.75
0.89
72.75
2.25
1.16
4.00
30.4
0.02
30.5
7.02
0.30
57.29
0.59
0.21
3.96
30.4
5.11
35.5
4.63
0.31
66.36
0.57
0.27
4.50
43.7
11.76
52.3
7.69
0.66
72.77
1.06
0.68
3.50
11.7
0.00
17.1
1.44
0.04
60.0
0.23
0.07
Result of
t testa
∗
∗
NS
∗
∗
∗
NS
∗
∗
Significant differences: ∗ P < 0.001; NS, non-significant differences.
Table 3. Antiradical and antibrowning activities of honeys from different sources
Antibrowning activity (%)a
Apple juice
Honey type (number
of samples)
Radical-scavenging
activity (%)a
Minced appleb
Spectrophotometric
analysis
Colorimetric
analysis
Nectar (n = 19)
Honeydew (n = 17)
28.7 ± 16.6
66.8 ± 18.1
33.7 ± 27.0
57.9 ± 32.2
36.3 ± 13.9
47.8 ± 15.7
33.2 ± 22.0
40.5 ± 22.9
a
b
Values are mean ± standard deviation of three replicates for each sample.
Values obtained with 2% honey per homogenate.
after the honey and DPPH solutions had been mixed.
This procedure without xylene allowed us to reduce
the solvent requirement and simplify the analytical
method.
The absorbance of each honey sample and the
minimum absorbance, when the radical has been
completely scavenged by the antioxidant, must be
taken into account. The results obtained for nectar
and honeydew honeys are summarised in Table 3.
Higher radical-scavenging capacity was observed for
honeydew honeys, in accordance with recent studies
showing that the darkest-coloured honey presents
the highest antioxidant capacity.13,25 Relationships
between antioxidant capacity and colour and/or other
physicochemical parameters of honey are discussed
below. Although medium–low standard deviations
were obtained in the triplicate antiradical assays
(from 0.1 to 6%), high variability was observed when
different honeys of the same botanical origin were
evaluated, which could be due to the fact that these
honeys were obtained from different beekeepers and
in some cases from different geographical regions.13
Inhibition of enzymatic browning
Honeys of different botanical origin were evaluated
as browning inhibitors in minced apple and apple
juice. The antibrowning activity of honeys on
fresh apple homogenates was assayed with five
honey concentrations ranging from 0.5 to 4% per
homogenate, after 1 h of incubation. Table 3 shows
the results expressed as % browning inhibition by
1072
honey solutions. In general, antibrowning activities
in both homogenates and juices were lower with
nectar honeys, mainly echium, rosemary and heather
honeys, than with honeydew honeys. When different
honey concentrations were added to minced apple, an
increase in antibrowning activity with concentration
was observed, but the effects did not follow a
standard pattern (Fig. 1). Because of the dark colour
of some higher-concentration honey solutions, only
values obtained with intermediate concentrations were
treated statistically. In these medium-concentration
honey solutions, colour interference was minimised
and the antibrowning effect was observable.
A significant positive correlation was found (r 2 =
0.633, P < 0.001) between the spectrophotometric
and colorimetric methods developed to evaluate the
antibrowning effect of honey in apple juice. When the
results obtained by these two methods were compared
with the honey antibrowning effect on minced
apple, the best correlation values were obtained with
P < 0.005 (r 2 = 0.448) for the spectrophotometric
method and P < 0.05 (r 2 = 0.343) for the colorimetric
method.
Relationship between antioxidant activity of
honeys and their physicochemical properties
Correlations between antioxidant content and colour24
and between antioxidant activity and total polyphenol content13,14 have been described previously for
some types of honey. In our study the evaluation
of 36 Spanish honeys showed a high correlation
J Sci Food Agric 87:1069–1075 (2007)
DOI: 10.1002/jsfa
0
1
2
3
4
5
100
90
80
70
60
50
40
30
20
10
0
0
2
1
3
4
browning inhibition (%)
100
90
80
70
60
50
40
30
20
10
0
browning inhibition (%)
browning inhibition (%)
Antioxidant capacity of Spanish honeys
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
% heather honey (w/w)
% honeydew honey (w/w)
% viper's bugloss honey (w/w)
0
Figure 1. Effect of honey concentration on browning inhibition of freshly minced apple.
Table 4. Pearson correlation coefficients of different assays versus physicochemical characteristics
Honey activity
Enzymatic browning inhibition
Minced apple
pH
Net absorbance
Acidity
Electrical conductivity
Polyphenol content
Apple juice
Radical-scavenging
effect
Honey 2%
Honey 3%
Spectrophotometric method
Colorimetric method
0.689∗∗
0.658∗∗
0.689∗∗
0.888∗∗
0.706∗∗
0.102
0.570∗∗
0.657∗∗
0.319
0.347∗
0.083
0.526∗∗
0.605∗∗
0.335∗
0.370∗
0.092
0.592∗∗
0.596∗∗
0.384∗
0.393∗
0.040
0.310
0.291
0.173
0.219
Significant differences at ∗∗ P < 0.001 or ∗ P < 0.05.
of radical-scavenging capacity with total polyphenol content and net absorbance (colour parameter).
Moreover, a positive correlation was also obtained
with honey pH, acidity and electrical conductivity
(Table 4). Hence the electrical conductivity, a parameter closely related to the concentration of minerals,
organic acids and proteins,40 accounted for >75%
of the variance in the radical-scavenging capacity
of honey (r 2 = 0.788), whereas the other parameters showed positive correlations with r 2 < 0.500.
Therefore the correlation obtained between antioxidant capacity and total polyphenol content suggests
that the phenolic compounds are partly responsible for
the antioxidant effects of honey, but obviously there
are other factors involved.
When honey antibrowning activities were studied statistically, the strongest linear relationship was
found between the parameters acidity and colour (P <
0.001). On the other hand, conductivity and polyphenol content only showed significant differences at P <
0.05, and no correlation was found with pH (Table 4).
According to these results, a simple expression
(Eqn (1)) based on electrical conductivity (EC),
acidity (AC) and net absorbance (NA) allowed the
estimation of the radical-scavenging capacity of honey
(Fig. 2A). However, when the same parameters were
used to estimate the antibrowning activity of honey,
the linear correlation was not as good (r 2 < 0.52;
Figs 2B and 2C).
Radical-scavenging activity
= 65.94EC + 0.48AC − 9.79NA − 16.78 (1)
J Sci Food Agric 87:1069–1075 (2007)
DOI: 10.1002/jsfa
When polyphenol content (instead of colour) was used
with electrical conductivity and acidity, the expression
obtained showed a good linear correlation, though
slightly lower than that obtained using net absorbance.
Separate evaluation of nectar and honeydew honeys
showed that the net absorbance and acidity of
nectar honeys were directly correlated with their
antioxidant capacity, whereas honeydew honeys
only showed relationships between radical-scavenging
activity and electrical conductivity and between
browning inhibition in minced apple and total honey
acidity (data not shown). The lack of correlation
between antioxidant capacity and colour for honeydew
honeys could be explained by their narrow range of
colour.
In conclusion, honeydew honeys showed higher
antioxidant capacities as radical-scavenging and
antibrowning agents than did nectar honeys. Honeydew honeys were generally characterised by higher pH,
electrical conductivity and acidity and darker colour
than nectar honeys.
ACKNOWLEDGEMENTS
This work was supported by research grant CAL02012 from INIA (MCYT, Spain). LV thanks IMIDRA
(Comunidad de Madrid) for her research grant.
RAP thanks the Spanish Ministerio de Ciencia y
Tecnolog ı́a for her contract from the Ramón y Cajal
programme.
1073
L Vela, C de Lorenzo, RA Pérez
y = -16.78+65.94EC+0.48AC-9.79NA
r2 = 0.822
Scavenging activity (%)
100.00
80.00
60.00
40.00
20.00
0.00
1
0
-1
Physicochemical parameters
-2
(a)
2
Antibrowning activity (%)
100.00
80.00
60.00
40.00
y =-4.16-27.02EC
+1.23AC+57.56NA
r2 = 0.517
20.00
0.00
-2
-1
(b)
Antibrowning activity (%)
100
1
2
0
Physicochemical parameters
3
y = 6.09-6.27EC+0.78AC+15.98NA
r2 = 0.491
80
60
40
20
0
-2
(c)
-1
1
0
2
Physicochemical parameters
3
Figure 2. Correlations between antioxidant activities of honeys and
their physicochemical characteristics (electrical conductivity (EC),
acidity (AC) and net absorption (NA)): A, DPPH radical-scavenging
activity; B, honey (2%) antibrowning activity in minced apple; C,
honey (4%) antibrowning activity in apple juice.
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