S2.0 S0308814613009618 Main [627769]

Analytical Methods
Suitability of antioxidant capacity, flavonoids and phenolic acids
for floral authentication of honey. Impact of industrial thermal treatment
Isabel Escrichea,⇑, Melinda Kadarb, Marisol Juan-Borrásb, Eva Domenecha
aInstitute of Food Engineering for Development, Food Technology Department, Universitat Politècnica de València, P.O. Box 46022, Valencia, Spain
bInstitute of Food Engineering for Development, Universidad Politécnica de Valencia, P.O. Box 46022, Valencia, Spain
article info
Article history:
Received 1 April 2012Received in revised form 20 September2012
Accepted 7 July 2013
Available online 15 July 2013
Keywords:
Honey
FlavonoidsPhenolic acidsAntioxidant capacityAuthenticationabstract
Total antioxidant activity, physicochemical parameters, and the profile of flavonoids and phenolic acid
compounds were evaluated for: their ability to distinguish between the botanical origins of four types
of Spanish honey, the impact of industrial thermal treatment, and the effect of the year of collection. Cit-
rus honey had the lowest levels of all the analysed variables, then rosemary and polyfloral, and honeydewthe highest ones. Botanical origin affects the profile of flavonoids and phenolic compounds sufficiently to
permit discrimination thanks to the predominance of particular compounds such as: hesperetin (in citrus
honey); kaempferol, chrysin, pinocembrin, caffeic acid and naringenin (in rosemary honey) and myrice-tin, quercetin, galangin and particularly p-coumaric acid (in honeydew honey). The impact of industrial
thermal treatments is lower than the expected variability as a consequence of the year of collection,
though neither factor has enough influence to alter these constituent compounds to the point of affectingthe discrimination of honey by botanical origin.
/C2112013 Elsevier Ltd. All rights reserved.
1. Introduction
Botanical authentication is one of the most important issues in
honey quality control, as it directly determines the market price.
Regulatory authorities, the food industry, retailers and consumers
are interested in knowing the origin and quality of honeys. Usually,
the identification of the botanical source of honey is carried out by
the calculation of the percentage of pollen. However, this analysisis not the most suitable for some kinds of honey such as citrus,
since the amount of pollen present is generally small and very var-
iable as the maximum secretion of nectar does not coincide with
anther maturation ( Ferreres, Giner, & Tomas-Barberán, 1994 ).
Therefore, it could be interesting to authenticate honey botanic
source considering specific chemical compounds that are only
present in specific nectars (or secretions of plants in the case of
honeydew honey), and consequently in the corresponding hon-
ey.Amongst the different analytical possibilities: volatile com-
pounds ( Escriche, Visquert, Juan-Borrás, & Fito, 2009 ), metals,
proteins, organic acids ( Mato, Huidobro, Simal-Lozano, & Sancho,
2006 ), sugars ( Cavia, Fernández-Muiño, Huidobro, Alvarez, &
Sancho, 2009 ), flavonoids and other phenolic compounds have
been widely used as a tool for this purpose ( Baltrušaityte ˙, Riman-
tas-Venskutonis, & C ˇeksteryte ˙, 2007; Escriche, Kadar, Juan-Borrás,
& Domenech, 2011; Frankel, Robinson, & Berenbaum, 1998; Iurlina,Saiz, Fritz, & Manrique, 2009; Lachman, Orsák, Hejtmánková, &
Kovárová, 2010b; Schramm et al., 2003; Yao et al., 2004 ). This is
due to the fact that these compounds are stable non-volatile sec-
ondarymetabolites, which appear to be relatively unaffected by
environmental factors ( Ferreres, Giner, & Tomas-Barberán, 1994 ).
For this reason, some of these compounds have been suggested
as ‘‘markers’’ in the determination of a specific honey: kaempferol
in rosemary honey ( Tomás-Barberán, Martos, Ferrerer, Radovic, &
Anklam, 2001 ), abscisic acid in heather honey ( Ferreres, Andrade,
& Tomás-Barberán, 1996 ), hesperetin in citrus honey ( Ferreres,
Garcia-Viguera, Tomás-Lorente, & Tomás-Barberán, 1993 ), luteolin
in lavender, quercetin in sunflower honey ( Yao et al., 2003 ).
However, it is important to emphasise that honey is not normally
commercialised in its raw state; on the contrary it usually under-
goes an industrialisation process. This is mainly because consumers
demand a fluid, non-crystallised product and although recently har-
vested raw honey is in a liquid state, it crystallises with greater or
lesser speed. Crystallisation depends on numerous factors such as
origin (botanical and geographical), temperature, moisture content,
and sugar content ( Cavia et al., 2009 ). Industrial manufacturing of
honey includes two stages that involve thermal treatments: lique-
faction (approx. 55 /C176C) to ensure that it is sufficiently liquid to han-
dle; and pasteurisation (approx. 80 /C176C) to destroy yeast that can
cause unwanted fermentation during the product’s shelf-life and
dissolve the crystallisation nuclei that cause honey to solidify,
ensuring that the honey stays in its liquid form for as long as possi-
ble ( Escriche, Visquert, Carot, Doménech, & Fito, 2008 ).
0308-8146/$ – see front matter /C2112013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.07.033⇑Corresponding author. Tel.: +34 963879146; fax: +34 963877369.
E-mail address: iescrich@tal.upv.es (I. Escriche).Food Chemistry 142 (2014) 135–143
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier .com/locate/foodchem

Heat processing has been extensively evaluated considering the
impact on the different components of honey such as: enzymes,
sugars and volatile compounds ( Escriche, Visquert, Juan-Borrás,
and Fito, 2009 ). However, the effect of industrial processing on
the antioxidant capacity of honey has not been studied in depth.A-
mongst the few works dealing with this topic, the work of Turk-
men, Sari, Poyrazoglu, and Velioglu (2006) should be mentioned.
They observed that heat treatment increases the antioxidant
capacity of honey, resulting in a positive effect on human health
as a consequence of these Maillard action products, but also a neg-
ative one, since browning caused by heating is not desirable by
consumers. Concerning this issue, it is important to highlight the
paper of Wang, Gheldof, and Engeseth (2004) who observed that
the impact of traditional processing on honey antioxidant capacity
(determined by oxygen radical absorbance) varies depending onthe type of honey. Buckwheat honey was more affected by process-
ing than clover honey in terms of reduction in antioxidant capacity.
In the same way, these authors showed that the impact of heat
processing on the phenolic profile was complex; some compounds
like quercetin and galangin only increased significantly in clover
honey.
For the above mentioned reasons, the present work aims to
determine to what extent flavonoids, phenolic compounds and
the antioxidant capacity of honey can be used in the authentication
of the botanical origin of four types of Spanish honey, and to eval-
uate the impact of industrial thermal treatment on this process. In
order to guarantee the reliability of the chromatographic proce-
dure used to quantify the flavonoids and phenolic compounds it
was validated before analysing the samples.
2. Materials and methods
2.1. Honey samples and their classification
Four types of Spanish honey (harvested in 2008 and 2009):
three of floral origin (citrus, rosemary and polyfloral) and one from
honeydew (forest origin) were used in this study. The botanical ori-
gin of the samples was ascertained by melissopalynological analy-
sis (Table 1 ). Conductivity is also given in this table for honeydew
in order to supplement the pollinic information for this type of
honey as required ( Council Directive, 2002 ). These honeys are the
most consumed in Spain. For each type of honey, and for each year,
4 different raw batches (15 kg each) were obtained directly from
local beekeepers (to ensure freshness). Each batch was divided into
3 parts; one was analysed in its raw state (unheated) and the other
2 were analysed following heat treatment (liquefaction and lique-
faction plus pasteurisation). Industrial liquefaction and pasteurisa-
tion processes were taken into account when choosing the
treatment temperatures. Accordingly, the liquefaction samples
were placed in a temperature-controlled oven (Selecta model
20000207 80L, Barcelona, Spain) at 45 ± 1 /C176C for 48 h, and the pas-
teurisation samples (previously liquefied) were heated at
80 ± 0.05 /C176C for 4 min in a temperature-controlled oil bath (Digi-
term 200, Selecta, Barcelona, Spain). The honey was pumped, using
a variable speed peristaltic pump (Heidolph model Pumpdrive
5001, Schwabach, Germany), through silicone tubing (6 mm bore
and 1 mm thickness) in the bath. Given that not all the honeys
had the same viscosity, the pump speed was adjusted in each case
to achieve the desired pasteurisation time. After thermal treat-
ments, all samples were quickly cooled to 30 /C176C.
2.2. Standards and reagents
All the standards (purity higher than 99%): hesperetin, naringe-
nin, caffeic acid, chrysin, p-coumaric acid, galangin, pinocembrin,kaempferol, myricetin and quercetin, were purchased from Extra-
synthese (Lyon Nord 69726 France). Amberlite XAD-2 resin (pore
size 9 nm and particle size 0.3–1.2 mm) was bought from Supelco
(Bellefonte, PA, USA), ABTS [2,2-azino-bis (3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt] was from Fluka Chemie, Buchs,
Germany. Stock solution of ABTS (2 mM) was prepared by dissolv-
ing in 50 mL of phosphate buffered saline (PBS), pH of the solution
was adjusted to 7.4 with 0.1 M NaOH. The solvents used for the
chromatography column were deionised water and analytical
grade hydrochloric acid and methanol. For HPLC analysis, metha-
nol and formic acid HPLC grade and bidestillated water were used,
purchased from VWR (Darmstadt, Germany). The membrane filter
(0.45 lm pore size) was acquired from Sartorius (Stedim Biotech,
Germany).
2.3. Melissopalynological analysis
The analysis and quantification of pollen were carried out fol-
lowing the recommendations of the International Commission for
Bee Botany ( Von Der Ohe, Persano, Piana, Morlot, & Martin,
2004 ). A light microscope (Zeiss Axio Imager, Göttingen, Germany)
at a magnification power of /C2400 with DpxView LE image analysis
software attached to a DeltaPix digital camera was used.
2.4. Physicochemical analysis
Physicochemical parameters [diastase activity, 5-hydroxymeth-
ylfurfural content (HMF), moisture content and electrical conduc-
tivity] were analysed following the recommendation of the
Harmonised Methods of the European Honey Commission
Bogdanov, (2002). Colour was determined using a millimetre Pfund
scale and a spectrocolorimeter (Minolta CM-3600d, Osaka, Japan).
Coordinates CIE L⁄a⁄b⁄were obtained from R1between 400 and
700 nm for D65 illuminant and 2 /C176observer. All tests were per-
formed in triplicate.
2.5. Analysis of flavonoids and phenolic acids
2.5.1. Extraction
The extraction of flavonoids and phenolic acids was carried out
as described by Baltrušaityte ˙et al. (2007) : ‘‘Sixty grams of amber-
lite resin were soaked in methanol for 10 min, then, most of meth-
anol was decanted and replaced by distilled water. The mixture
was stirred, allowed to stand for 5–10 min and was packed into a
glass column (25 /C22 cm). Honey samples (25 g) were thoroughly
mixed with 250 mL of distilled water and adjusted to pH 2 with
concentrated HCl. The solution was slowly filtered through the col-
umn, which was washed with 250 mL of acidified water (pH 2 withHCl) and subsequently rinsed with 300 mL of neutral distilled
water to remove all sugars and other polar compounds of honey.
The flavonoids and phenolic compounds were eluted from the sor-
bent with 250 mL of methanol. The extract obtained was concen-
trated under vacuum at 40 /C176C in a rotary evaporator (Laborota
4000/HB/G1, Heidolph Instruments GmbH, Schwabach, Germany).
The residue was dissolved in 5 mL of distilled water and extracted
three times with 5 mL of diethyl ether. The extracts were combined
and the solvent was removed by flushing with nitrogen. The dried
residue was then redissolved in 1 mL of methanol (HPLC grade)
and filtered through a membrane filter. Three replicate extractions
were performed for each sample.
2.5.2. HPLC analysis
Analyses of the extracts were carried out by HPLC [Alliance
2695, with a 2996 photodiode array detector (Waters, USA)]. Flavo-
noids and phenolic compounds were separated on a Sunfire TM
analytical C18 column, 150 /C24.6 mm i.d. and particle size 5 lm136 I. Escriche et al. / Food Chemistry 142 (2014) 135–143

(Waters, Ireland). The binary mobile phase consisted of solvent A
(water and formic acid, 95:5) and solvent B (100% methanol).
According to the method of Martos et al. (2000) , the following gra-
dient was used: starting with 30% methanol (B) which was kept
isocratically for 15 min, and then followed by a gradient to obtain
40% methanol at 20 min, 45% methanol at 30 min, 60% methanol at
50 min, and 80% methanol at 52 min. Finally, isocratic elution with
80% methanol was carried out until 60 min. The flow rate was
1 mL/min and the injection volume was 30 lL. Chromatograms
were recorded at three wavelengths (290, 340 and 370 nm). Flavo-
noids and phenolic acids were identified by comparison of chro-
matographic retention times and UV spectral characteristics of
unknown analytes with authentic standards and available litera-
ture data ( Merken & Beecher, 2000 ). Calibration curves were con-
structed via least squares linear regression analyses of the ratioof the peak area of each representative compound versus the
respective concentration. Quantitative results were expressed as
mg of compound per 100 g of honey.
2.5.3. Total antioxidant activity: ABTS
+radical cation decolourisation
assay
The antioxidant activity of honey samples was determined by
the reaction with stable ABTS+radical cation according to the
method described by Baltrušaityte ˙et al. (2007) . ABTS+was pro-
duced by reacting 50 mL of ABTS stock solution with 200 lLo f
70 mM K 2S2O8. The mixture was left to stand in the dark at room
temperature for 15–16 h before use. For the evaluation of antioxi-
dant activity, the ABTS+solution was diluted with PBS to obtain an
absorbance of 0.800 ± 0.030 at 734 nm. Ten microlitres of honey
phenolic extract solution, obtained as was described in Section
2.5.1., were mixed with 3 mL of ABTS+solution. The absorbance
was read at room temperature after 1, 4, 6 and 10 min. PBS solution
was used as a blank. The measurement was performed in triplicate.
The percentage decrease of the absorbance at 734 nm was calcu-
lated by the formula I=[ (A-AA)/AB]/C2100; where: I= ABTS+inhibi-
tion %; AB= absorbance of a blank ( t= 0 min); AA= absorbance of a
tested honey extract solution at the end of the reaction
(t= 10 min).
2.5.4. Validation of the analytical method (HPLC–DAD) for flavonoids
and phenolic acids
Due to the fact that honey has a complex matrix, it is necessary
to validate the methods used to analyse its components. This fact is
especially important for those compounds that have very low con-
centration levels such as flavonoids and phenolic acids, in order to
ensure suitable levels of recovery and repeatability.
The validation parameters: linearity, accuracy and precision
(‘‘repeatability’’: intraday precision and ‘‘reproducibility’’: interdayprecision), were obtained according to the criteria described by the
Commission Decision (2002 ) and by ICH (1996) . The sensitivity of
the method was proved with the limit of detection (LOD) and the
limit of quantification (LOQ) of the diferents analytes.
Linearity was evaluated using a matrix similar to honey (com-
mercial glucose syrup, Roquette, España, S.A.) as a blank. This
was fortified at 4 final concentration levels (0.1, 0.2, 0.6 and
1.2 mg/100 g) and submitted to the same extraction procedure as
the honey ( n= 4). Linear calibration curves were constructed from
the peak areas of the compounds versus the correspondent concen-
trations of the fortified blanks. The accuracy of each flavonoid and
phenolic acid was evaluated with the recovery test using the same
level concentrations as linearity (0.1, 0.2, 0.6 and 1.2 mg/100 g),
(n= 3).
The precision of the method was expressed as the relative stan-
dard deviation (RSD), in this way the repeatability was calculated
from the analysis of six blank samples fortified at each of the four
specified levels of fortification, and performed by the same opera-tor on the same day. To evaluate the reproducibility the analyses
were repeated on three consecutive days. LODs and LOQs were
estimated by fortifying blanks at different concentrations and
applying the extraction procedure, and were determined as the
amount of analyte for which signal-to-noise ratios ( S/N) were high-
er than 3 and 10, respectively. The stock standard solution of the all
the compounds was prepared by dissolving the appropriate
amounts in methanol. This stock solution was stored at /C020/C176C.
2.6. Statistical analysis
A multifactor analysis of variance (ANOVA) (using Statgraphics
Plus version 5.1.) was carried out to study the influence of the type
of honey, the thermal treatments applied and the year of harvest-
ing on the physicochemical parameters and on the flavonoids and
phenolic compounds of honey. Three factors were taken into con-
sideration: the type of honey (citrus, rosemary, polyfloral and hon-
eydew), the thermal treatment applied (liquefaction/
pasteurisation) and the year (2008/2009). The double interactions
between these factors were also considered. The method used for
multiple comparisons was the LSD test (least significant difference)
with a significance level a= 0.05. In addition to this, the flavonoids
and phenolic compounds data were analysed using a PrincipalComponent Analysis (PCA) applying the software Unscrambler
X.10.
3. Results and discussion
3.1. Melissopalynological and physicochemical characterisation of
honey
The samples used in this study were accurately classified as
belonging to a specific botanical variety according to the melissop-
alynological characterisation, the most reliable and useful way of
determining the botanical origin of honey.
The percentage of pollen from the principal botanical species
varied notably according to the origin of the honey. For citrus hon-
ey this percentage was between 15% and 37% and for rosemary
honey it ranged between 15% and 31%. In the case of honeydew
honey, dew elements between 1.9% and 2.5% were found. The hon-
eys were considered to be polyfloral, if the percentage of a specific
type of pollen was not enough for classification as monofloral.
Table 2 shows the average values resulting from the multifactor
analysis of variance (ANOVA), including the F-ratio, obtained for
the physicochemical parameters (diastase activity, HMF, moisture
content, electrical conductivity, colour Pfund, CIEL ⁄a⁄b⁄coordi-
nates) and TAA (Total Antioxidant Activity).
Diastase activity and electrical conductivity were very different
depending on the botanical origin of honey as previously shown by
other authors ( Escriche et al., 2011 ). All samples fulfilled the min-
imum required level established by the European Directive ( Coun-
cil Directive, 2002 ), as in all cases diastase activity was well over
800. As expected, honeydew honey had the highest levels of dia-
stase activity and conductivity. Citrus and rosemary honeys had
considerably lower diastase activity average values (8.53 and
11.74) and electrical conductivity (0.17 and 0.22) than the polyfl-
oral and honeydew types, with values of 19.74 and 20.54 for dia-
stase and 0.37 and 1.00 for conductivity. Other authors have
shown that amongst floral honeys, citrus and rosemary types have
relatively low conductivity levels ( Corbella & Cozzolino, 2006; Ter-
rab, Gonzalez, Diez, & Heredia, 2003 ). As expected, polyfloral hon-
eys had intermediate values, given that they come from many
different types of nectar ( Corbella & Cozzolino, 2006 ). The HMF
content of raw samples was always quite low in this study, the
only exceptions being some rosemary samples with values up toI. Escriche et al. / Food Chemistry 142 (2014) 135–143 137

15.6 mg/kg, which demonstrates that almost all the raw honey was
fresh. The values of HMF increased a little with the heat treat-
ments, reaching 7.65 mg/kg average value in liquefied samples
and 8.18 mg/kg in pasteurised samples. In the same way, and as
a consequence of the thermal treatment, the average value of dia-
stase activity decreased slightly from 16.07 (ID) in the raw samples
to 13.80 in the pasteurised ones. This logical behaviour is related to
the implication of both parameters as indicators of freshness.
Moisture content in all samples met the requirements of the
Council Directive of 2002 (maximum limited to 20 g/100 g in order
to avoid yeast fermentation), as the maximum value did not exceed
17 g/100 g.
The higher the F-ratio (quotient between variability due to the
considered effect and the residual variance), the greater the effect
that a factor has on a variable. According to this, conductivity, vari-
ables related to colour (colour Pfund, and CIEL⁄a⁄b⁄coordinates),
HMF diastase, and ATT were most affected by the type of honey;
whereas moisture was most affected by treatment. Honeydewhoney presented the greatest values of both antioxidant capacities
and several physicochemical parameters when compared to nectar
honeys. These results are in accordance with those reported by
Vela, De Lorenzo, and Perez (2007) for Spanish honeys.
The factor ‘‘year’’ has an influence on moisture and diastase
activity but above all on conductivity. The highest number of sig-
nificant interactions was found for the combination of factors hon-
ey and year. This significant interaction indicates that the value of
these parameters (conductivity, moisture, Pfund colour scale, TAA
and diastase) is different from year to year depending on the type
of honey. In the case of ATT the interaction between type of honey
and treatment was also significant. Wang et al. (2004) observed
that the impact of traditional thermal processing on honey antiox-
idant capacity varies depending on the type of honey, due to the
complicated chemical composition (pigments, phenolic com-
pounds, enzymes, Maillard reactions and other minor compounds)
which varies between honeys from different floral sources
(Baltrušaityte, Rimantas-Venskutonis, and Ceksteryte, 2007 ;Saxena, Gautam, & Sharma, 2010 ). In this work, thermal treatment
caused a significant increase of ATT (from 59.34% inhibition in the
raw sample to 63.18% in the pasteurised) as expected, as the lique-
faction and pasteurisation temperatures applied are not very high
(Turkmen et al., 2006). In the same way, the difference between
years for TAA was significant. However, it is important to empha-
sise that although treatment and year were significant, the factor
‘‘botanical origin’’ had the greatest influence on the antioxidant
activity.
As other authors suggest, the antioxidant activity of different
kinds of honeys can be related to specific physicochemical param-
eters and colour ( Baltrušaityte ˙et al., 2007; Gonzalez-Miret, Terrab,
Hernanz, Fernandez-Recamales, & Heredia, 2005 ). The possible
correlations between all of these parameters were checked in this
work using Pearson correlation coefficients ( r).Table 3 shows the
correlation matrix obtained for each pair of variables. The number
in brackets is the P-value which tests the statistical significance of
the estimated correlations at the 95.0% confidence level. As ex-pected, there was no correlation between HMF or moisture and
the rest of the analysed parameters. However, Aljadi & Kamaruddin
(2004) show that Malaysian floral honeys with higher water con-
tent had higher antioxidant capacity. Good correlations were ob-
served in this work between all the parameters related to colour
(especially with regard to Pfund scale), and conductivity, diastase
activity and TAA. The correlation between colour and conductivity,
colour and ID is widely accepted ( Perez, Iglesias, Pueyo, Gonzalez,
& De Lorenzo, 2007; Saxena et al., 2010 ). Different authors showed
that the colour intensity of honey is related to pigments such as
carotenoids and flavonoids, and therefore, pigments have a role
in the antioxidant activities of the honey samples ( Saxena et al.,
2010 ).Estevinho, Pereira, Moreira, Dias, & Pereira (2008) demon-
strated that dark honeys from Northeast Portugal have a signifi-
cantly higher level of antioxidant capacity than the clear ones.
Escuredo, Silva, Valentao, Seijo, and Andrade (2012) observed that
colour in Rubus honeys presented a significant correlation with the
total phenol content and some individual phenolic compounds.Table 1
Percentage of pollen for the most abundant botanical species in the four batches analysed for 2008 and 2009 for each type of honey. For honeydew, conduc tivity is also given in
order to supplement the pollinic information.
Batch1 Batch2 Batch3 Batch4
Citrus
(2008)21% Citrus sp., 2% Rosmarinus
officinalis ,Genista sp.,Diplotaxis
erucoides , and others17% Citrus sp., 11% Rosmarinus
officinalis ,Diplotaxis erucoides , and
others15% Citrus sp., 8% Diplotaxis
erucoides ,Prunus dulcis ,Vitis
vinifera , and others19% Citrus sp., Labiatae, Erica sp.,
Salix sp., Hypecoum imberbe , and
others
Citrus
(2009)22% Citrus sp., 15 % Prunus dulcis ,
Ceratonia siliqua , and others18% Citrus sp.,Taraxacum sp.,
Quercus sp.Labiadas , and others37% Citrus sp.,Taraxacum sp.,
Genista sp.,Labiadas , and others28% Citrus sp., type Taraxacum ,
Helianthus annuus ,Erica sp., and
others
Rosemary
(2008)25% Rosmarinus officinalis , 12%
Diplotaxis sp., and others15% Rosmarinus officinalis , 17%
Diplotaxis sp., and others21% Rosmarinus officinalis , 17%
Prunus sp.,Thymus sp.,Genista sp.,
and others19% Rosmarinus officinalis , 12%
Hypecoum imberbe ,Thymus sp., and
others
Rosemary
(2009)30% Rosmarinus officinalis ,Genista sp.,
Erica sp.,Prunus dulcis , and others23% Rosmarinus officinalis , 18%
Thymus sp.,Satureja montana , and
others31% Rosmarinus officinalis ,8 %
Diplotaxis sp.,Thymus sp., and
others25% Rosmarinus officinalis , 21%
Prunus dulcis ,Diplotaxis sp., and
others
Polyfloral
(2008)23% Echium sp., 4% Helianthus annuus ,
Lavandula stoechas , and others39% Echium vulgare , 16% Helianthus
annuus , 11% Lavandula , and others26% Echium vulgare , 21%
Eucalyptus sp., 10% Lavandula , and
others31% Echium sp., 14% Eucalyptus sp.,
8%Helianthus annuus , and others
Polyfloral
(2009)20% Anthyllis cytisoides, 14%
Helianthus annuus , 15% Umbelliferae ,
and others27% Rubus sp., 19% Diplotaxis sp.,
Olea europea ,Crataegus monogyma ,
and others19% Anthyllis cytisoides , 18%
Echium vulgare , 9% Prunus dulcis,
and others24% Salix sp.,16% Rosaceae , 10%
Rosmarinus officinali s, and others
Honeydew
(2008)HDE = 1.9
55% Castanea sativa , 11% Rubus sp., 4%
Lavandula stoechas ,Erica sp., and
othersHDE = 2.0
27% Erica sp, 18% Castanea sativa,
10% Lavandula stoechas, and othersHDE = 2.5
31% Echium sp., 14% Eucalyptus sp.,
8%Helianthus annuus , and othersHDE = 2.3
43% Echium sp., 4% Helianthus
annuus , Lavandula stoechas, and
others
Conductivity = 1.150 mS/cm Conductivity = 0.892 mS/cm Conductivity = 0.884 mS/cm Conductivity = 0.900 mS/cm
Honeydew
(2009)HDE = 1.9
16% Rubus sp., 6% Thymus sp.,
Cruciferae, Onobrychis viciifolia , and
othersHDE = 1.0
26% Rubus sp., 7% Calluna vulgaris ,
11% Erica sp., and othersHDE = 2.2
21% Rosaceae, 8% Thymus sp.,
Quercus sp., Crucíferae, Salix sp.,
and othersHDE = 2.0
9%Thymus sp. 9% Rosaceae, Vicia
faba, Helianthus annuus, Rubus sp.,
and others
Conductivity = 0.893 mS/cm Conductivity = 1.390 mS/cm Conductivity = 0.891 mS/cm Conductivity = 0.896 mS/cm138 I. Escriche et al. / Food Chemistry 142 (2014) 135–143

The contribution of specific phenolic compounds to the TAA was
reported to be quite significant by other authors ( Gheldof, Xiao-
Hong, & Engeseth, 2002 ). Therefore, in this study it was considered
appropriate to evaluate some of these specific antioxidant com-
pounds, such as flavonoids and phenolic acids, in depth.
3.2. Identification of flavonoids and phenolic acids
Considering the four types of honey submitted to thermal treat-
ments, and the two different years, 8 flavonoids and 2 phenolic
acids were found. The phenolic acids (caffeic acid and p-coumaric
acid) were quantified at 340 nm. The flavonoids were quantified
at 290 nm (naringenin and hesperetin), at 340 nm (pinocembrin,
chrysin and galangin) and at 370 nm (myricetin, quercetin and
kaempferol). In addition, for identification the following kwere
considered: 244, 323 (caffeic acid); 309 ( p-coumaric acid); 289
(naringenin); 239, 286 (hesperetin); 290 (pinocembrin); 267, 313
(chrysin); 221, 265, 310, 358 (galangin); 233, 252, 367 (myricetin);
226, 254, 368 (quercetin); 265, 364 (kaempferol).
3.3. HPLC–DAD validation method
Table 4 shows the validation parameters of the method carried
out in this work to quantify flavonoids and phenolic acids in honey.
Good linearity was found as r2values ranged from 0.993 for galan-
gin to 0.999 for hesperetin, naringenin, and quercetin. The only
exception was myricetin whose r2was worse than the rest (0.986).
Considering the results of the recovery test, this method can be
considered accurate, except for caffeic acid, chrysin and myricetin
since for these compounds there are values for different levels that
lie outside the range (70–120%) accepted by the Commision Deci-
sion (2002) .The level 0.2 mg/100 g for caffeic acid (recovery 64%)
and the level of 1.2 mg/100 g for chrysin (123%) lies outside this
range. However, although these recoveries are not close to 100%,
they can be considered acceptable since they were reproducible
(Martinez Vidal et al., 2009 ). Poorer results were found for myrice-
tin, since for the two first concentration levels (0.1 and 0.2 mg/
100 g), the recoveries were far from the required values. In general,
the recoveries for all the compounds were higher than previously
reported by D’Arcy in 2005 , using Amberlite XAD-2 in the extrac-
tion step. He only reported acceptable recoveries for hesperetin
(88%) and chrysin (92%); on the contrary compounds like caffeic
acid, quercetin and p-coumaric acid showed unsatisfactory values:
22.57% and 38%.
Precision was evaluated by testing the repeatability (intraday
precision) and the reproducibility (interday precision). Table 4
shows the interday precision expressed as the relative standard
deviation in % (RSD). These values were lower than 20% for allthe compounds and concentration levels assayed, pinocembrin
and chrysin being the only exceptions, presenting values a little
higher than 20% for levels 0.2 and 0.6 mg/100 g, respectively. As
expected, the intraday precision was always less than the interday
precision. The values of LOQ (limit of quantification) ranged from
0.02 to 0.06 mg/100 g.
3.4. Flavonoids and phenolic compounds in honeys
Table 5 shows the flavonoids and phenolic compounds quanti-
fied in citrus, rosemary, polyfloral and honeydew honey, before
(R, raw) and after heat treatment ( L, liquefaction and P, pasteurisa-
tion). In addition, for each one of the analysed compounds, this ta-
ble shows the ANOVA F-ratio for the three factors considered (type
of honey, treatment and year) and their respective double interac-
tions. Most of the compounds are found in the four kinds of honey
but in different quantitative proportions. In fact, for all the quanti-
fied compounds, the ANOVA analysis shows significant differencesTable 2
Physico-chemical parameters, CIE L⁄a⁄b⁄colour and TAA (total antioxidant activity) analysed in honey samples ( Citrus , rosemary, polyfloral and honeydew) before and after heat treatment (R = raw; L = liquefaction and
P = pasteurisation) and year (2008 and 2009), and ANOVA F-ratio for each of the three factors (honey, treatment, and year) and their respective double interactions.
Honey factor ( H) Treatment factor ( T) Year factor ( Y) Factor interactions
Physicochemical parameters Citrus Rosemary Polifloral Honey-dew ANOVA
F-ratioR L P ANOVA
F-ratio2008 2009 ANOVA
F-ratioH⁄TT ⁄YH ⁄Y
Diastase activity (ID) 8.53c11.74b19.74a20.54a108***16.07a15.54a13.80b6*12.42b17.86a92***1.17ns3.46ns11**
HMF (mg/kg) 4.48c14.71a6.10b 3.91c151***6.08b7.65a8.18a10*9.10a5.50b78***1.72ns0.18ns2ns
Moisture (g/100g) 15.51b16.11a 15.03c15.31b32***16.67a14.98b14.97b 206***15.09b16.00a133***1.18ns171.79***22**
Conductivity (mS cm/C01) 0.17d0.22c0.37b1.00a11075***0.45a0.45a0.44b5ns0.33b0.55a37974***0.29ns0.48ns23528***
Pfund colour (mm) 15.50d42.33c 77.83b116.16a1410***61.25a63.37a64.25a2ns61.00b64.92a11*0.47ns2.56ns16**
CIE L⁄a⁄b⁄colour
L⁄47.99a39.20b34.32c26.02d576***37.89a36.41b36.36b7*36.86a36.90a0.01ns2.46ns0.33ns3ns
a⁄0.41d5.15c10.53a6.73b134***5.26a6.00a5.85a2ns5.79a5.62a0.21ns0.40ns0.14ns0.9ns
b⁄22.46a20.85ab17.27b6.12c46***16.09a17.06a16.88a0.3ns17.03a16.32a0.42ns0.07ns0.14ns1ns
TAA (% inhibition) 39.46c57.87b66.86b80.42a61***59.34ab56.68b63.18a4**63.27a56.20b12***4.10**0.19ns14***
For each factor, different letters in each row indicate significant differences at 95% confidence level as obtained by the LSD test.
ns = Not significant.
*p< 0.05.
**p< 0.01.
***p< 0.001.I. Escriche et al. / Food Chemistry 142 (2014) 135–143 139

between the four types of honey. This result demonstrates the
influence of the kind of honey on the quantity of these types of
compounds. Several reports show possible correlations between
floral origin and the flavonoid profile ( Yao et al., 2004 ). As ex-pected, prevalence of some individual components was observed
in this work. Specifically, the flavone hesperetin, was only found
in citrus honey (average level of 0.6 mg/100 g); in fact, it is consid-
ered as a marker compound for this variety of honey ( Ferreres,Table 3
Correlation matrix (Pearson correlation coefficients) between Total Antioxidant Activity (TAA), physicochemical parameters and colour.
TAA HMF ID Moisture Conductivity Colour Pfund L⁄a⁄
TAA (% inhibition)
HMF (mg/kg) 0.043
(0.768)
Diastase activity (ID) 0.601 /C00.370
(0.000) (0.011)
Moisture (g/100 g) /C00.195 -0.194 0.159
(0.182) (0.182) (0.276)
Conductivity (mS/cm/C01) 0.551 /C00.327 0.726 /C00.029
(0.000) (0.025) (0.000) (0.841)
Pfund colour (mm) 0.826 /C00.238 0.822 /C00.163 0.802
(0.000) (0.102) (0.000) (0.265) (0.000)
L⁄/C00.845 0.215 /C00.768 0.155 /C00.766 /C00.978
(0.000) (0.141) (0.000) (0.289) (0.000) (0.000)
a⁄0.648 /C00.001 0.774 /C00.152 0.707 0.724 /C00.716
(0.000) (0.994) (0.000) (0.299) (0.000) (0.000) (0.000)
b⁄/C00.756 0.337 /C00.643 -0.022 /C00.824 /C00.900 0.857 /C00.609
(0.000) (0.021) (0.000) (0.880) (0.000) (0.000) (0.000) (0.000)
Numbers in brackets = P-value at the 95.0%.
Table 4
Validation parameters of the analysed flavonoids and phenolic acids.
% Of Recovery and intraday precision (% RSD) Interday precision (%RSD) r2LOQ (mg/100g)
0.1 0.2 0.6 1.2 0.1 0.2 0.6 1.2
(mg/100g) (mg/100g)
Caffeic acid 94(10) 64(10) 90(9) 87(8) 11 12 11 11 0.998 0.05
Chrysin 87(10) 110(9) 114(8) 123(8) 12 22 10 14 0.996 0.02Galangin 118(12) 97(15) 94(10) 100(10) 17 11 11 10 0.993 0.02Hesperetin 115(11) 100(11) 99(6) 102(8) 13 12 9 9 0.999 0.05
Kaempferol 120(8) 118(7) 105(7) 118(6) 12 11 12 11 0.998 0.05
Myricetin 35(10) 20(8) 70(7) 80(8) 11 9 9 9 0.986 0.05Naringenin 120(9) 107(7) 111(7) 115(6) 11 12 12 9 0.999 0.06p-Coumaric 110(10) 107(12) 94(11) 108(9) 19 14 14 9 0.997 0.03
Pinocembrin 107(7) 93(6) 95(5) 102(8) 22 10 9 10 0.997 0.03Quercetin 119(9) 118(14) 112(11) 117(8) 10 16 17 12 0.999 0.05
Table 5
Flavonoids and phenolic compounds quantified in citrus, rosemary, polyfloral and honeydew honey, before and after heat treatment (R = raw; L = liquefac tion and
P = pasteurisation) and year (2008 and 2009); and ANOVA F-ratio for each of the three factors (honey, treatment and year) as well as their respective double interactions.
Flavonoids and phenolic
compounds (mg/100 g ofhoney)Honey factor ( H) Treatment factor ( T) Year factor ( Y) Interaction
Citrus Rosmary Polyfloral Honey-
dewANOVA
F-ratiooRLP ANOVA
F-ratio2008 2009 ANOVA
F-ratioH
⁄TH⁄YT⁄Y
Naringenin 1.3c2.3a2.1a1.8b32***1.9a1.9a1.9a0.6ns1.8a1.9a2ns1ns24***1ns
Hesperetin 0.6 n.d. n.d. n.d. n.d 0.5b0.8a0.7ab3ns0.7a0.6a3nsn.d n.d 5ns
Caffeic acid 0.6c2.3a1.8b1.6b62***1.5a1.6a1.6a0.2ns1.8a1.3b36***0.9ns40***0.4ns
p-Coumaric acid 0.4c2.8b2.9b31.4a21609***10.3a9.0b8.8b86***9.3a9.4a0.6ns73***3*0.1ns
Pinocembrin 0.8c2.7 ă 1.9b1.9b72***1.9a1.9a1.8a0.2ns1.8a1.9a2ns4**36***1ns
Chrysin 0.9d2.3a1.5c1.9b55***1.6a1.7a1.6a0.9ns1.6a1.7a0.9ns4**28***2ns
Galangin 0.3c1.2a0.8b1.4a62***1.0a0.9ab0.8b4*0.8b1.0a10**2ns9***0.04ns
Quercetin 0.3c0.5b0.4c0.9a43***0.6a0.5a0.6a0.4ns0.6a0.5b3ns4**2ns1ns
Kaempferol 0.7c2.7a1.2b0.4d669***1.5a1.2b1.2b20***1.4a1.2b16***40***14***0.9ns
Myricetin n.d.cn.d.c1.3b2.6a204***2.7a2.5a0.5b5⁄3.2a1.1a11**1.7ns26**1.6ns
Different letters in each row for each factor indicate significant differences at 95% confidence level as obtained by the LSD test.
ns = Not significant.n.d. = Not detected.
*p< 0.05.
**p< 0.01.
***p< 0.001.140 I. Escriche et al. / Food Chemistry 142 (2014) 135–143

et al., 1993; Soler, Gil, Garcia-Viguera, & Tomás-Barberan, 1995 ). In
other Spanish citrus honey samples this compound ranged be-
tween 0.07 and 0.76 mg per 100 g honey ( Escriche et al., 2011 ).
Kaempferol, described as a floral marker for rosemary honey
(Tomás-Barberán et al., 2001 ), was not only found in rosemary
honey (2.7 mg/100 g) in this work, but also in the other three types,
although in considerably lower amounts .The lowest average level
of kaempferol was observed in honeydew honey (0.4 mg/100 g).
The composition of phenolic acids also depends on the plant
source ( Lachman et al., 2010a ). In this work, the most abundant
compound found in all cases, with the exception of citrus honey,
was p-coumaric acid. Specifically, it is important to underline the
high average value of p-coumaric acid (31.4 mg/100 g) found in
the honeydew honey. In this work, this phenolic acid, together
with caffeic acid makes up: 17% in citrus, 30% in rosemary, 34%
in polyfloral and 74% in honeydew, of the total quantified com-
pounds in these types of honey. On the other hand, ellagic acid
and gallic acid were not detected in the studied honeys, despite
the fact they were widely found by other authors. For instance,
these acids were major compounds in Australian eucalyptus hon-
eys ( Yao et al., 2004 ). It has to be taken into account that the con-
centrations of the quantified compounds can vary a great deal, notonly due to the origin of the honey, but also with the conditions of
the analytic procedures used. In fact, D ´Arcy (2005) proved that
these kinds of compounds are not well retained in the amberlite
XAD-2 resin at the pH used. Consequently, the results are onlycompletely comparable if the same analytical method is applied.
With respect to thermal treatments, the ANOVA result demon-
strates that there is only a significant decrease for 4 compounds:
galangin, kaempferol myricetin and p-coumaric acid. However,
this diminution only seems important for myricetin, as a result
of pasteurisation. The factor ‘‘year’’ was only significant for 4 of
the 10 studied compounds (caffeic acid, galangin, kaempferol
and myricetin). The double interactions, between ‘‘type of honey’’
and ‘‘treatment’’, and ‘‘type of honey’’ and ‘‘year’’, were significant
for 5 of the 10 compounds, and 8 of the 10 compounds, respec-
tively. This significant interaction shows that the values of these
compounds change as a function of the treatment applied and
as a function of the year of analysis ( Fig. 1 ), according to the type
of honey.
Once the individual behaviour of each compound was studied, a
PCA was used to assess the overall effect of the type of honey, ther-
mal treatments and year on the flavonoids and phenolic com-
pounds. Fig. 2 shows the biplot of sample scores and compound
Fig. 1. Typical HPLC–UV chromatograms of flavonoids and phenolic acids in honeydew and rosemary honey.I. Escriche et al. / Food Chemistry 142 (2014) 135–143 141

loadings obtained by means of PCA analysis. Samples in Fig. 2 were
codified as (type of honey-thermal treatment-year). The first two
components explained 80% of the total variance (PC1 61% and
PC2 19%). It shows that the first principal component clearly differ-
entiates citrus honey located in the left quadrant from the honey-
dew honey and the rosemary one, both located in the right
quadrant. The polyfloral honey is placed in the middle of the plot.
The loadings of each compound on the principal components
clearly show that the grouping of the different types of honey is
primarily influenced by certain compounds. For instance, com-
pounds that are only found in citrus honey, such as hesperetin,
are mainly responsible for the difference between this honey and
the others. In the case of rosemary: kaempferol, chrysin, pinocem-
brin, caffeic acid and naringenin are the compounds that contrib-
ute most to its differentiation since these compounds are present
in greater quantities in this variety. The same occurs in honeydew
honey; since compounds as myricetin, quercetin, galangin and par-ticularly p-coumaric acid can be considered as essential in the dif-
ferentiation of this honey from the others. The results demonstrate
that flavonoids and phenolic compounds could be a good tool for
discriminating the floral origin of honey.
By focusing on a specific type of honey, and considering that
closeness between samples indicates similar behaviour in terms
of the analysed compounds, a greater difference was observed be-
tween years (2008 and 2009) than between the thermal treat-
ments applied (raw, liquefaction and pasteurisation). This similar
behaviour between samples, despite the thermal treatments, is
also observed when the physicochemical parameters (in this and
other works, Rand & Bosch-Reig, 1998 ) and the volatile fraction
are analysed ( Escriche, Visquert, Juan-Borrás, and Fito, 2009; Escri-
che et al. 2008 ). This is probably due to the fact that the treatments
applied (under moderate industrial conditions) are not aggressive
enough to affect the intrinsic properties of honey.
It is well known that properties of honeys from different years
vary depending on flowering and according to the collected nectar.Consequently certain active components of honey can be affected,
to a certain extent, by environmental factors changing from year to
year (rainfall, temperature, etc.) ( Yao et al., 2003 ). Although small
differences occur in the characteristics of honeys as a consequence
of the variation in climate conditions between years, they do not
seem important enough to notably modify the amount of flavo-
noids and phenolic compounds inherent to a type of honey. The
fact that the concentration of these compounds does not change
markedly due to the environmental and processing conditions is
especially relevant for monofloral honeys, since the presence of
some specific natural antioxidant compounds (effective in protect-
ing human health and reducing the risk of heart disease) may give
them an important added value.
4. Conclusions
Good Pearson correlations were observed between total antiox-
idant activity and colour, especially with regard to Pfund scale
(0.826) and L ( /C00.845), and some weaker ones with diastase activ-
ity (0.601) and conductivity (0.551). Honeydew honey had the
highest levels of all these parameters, rosemary and polyfloral
had intermediate values, and citrus the lowest. This study confirms
that the profile of flavonoids and phenolic compounds is affected
by the botanical origins of honeys to the point of contributing to
their differentiation. This is mainly due to the prevalence of some
specific compounds: hesperetin in citrus honey; kaempferol, chry-
sin, pinocembrin, caffeic acid and naringenin in rosemary honey
and myricetin, quercetin, galangin and particularly p-coumaric
acid in honeydew honey. In general terms, the impact of industrial
thermal treatments on this profile is less important that the logical
variability due to the year of harvesting (as a consequence of envi-
ronmental factors), which proves these processing conditions are
not aggressive enough to modify the intrinsic properties of honey
considering their botanical origin.
Fig. 2. PCA biplot of score samples (type of honey-thermal treatment-year) and compound loading (flavonoids and phenolic compounds).Type of honey (C ‘‘citr us’’, R
‘‘rosmary’’, P ‘‘polyfloral’’ and H ‘‘honeydew’’); thermal treatment (R ‘‘raw’’, L ‘‘liquefied’’and P ‘‘pasterised’’), year (8 ‘‘2008’’ and 9 ‘‘2009 ’’).142 I. Escriche et al. / Food Chemistry 142 (2014) 135–143

Acknowledgments
The authors acknowledge the financial support of the Valencian
Regional Ministry of Business Education and Science (project
CV2007-244) and the Universidad Politécnica de Valencia (project
PAID-05-08-3703).
References
Aljadi, A. M., & Kamaruddin, M. Y. (2004). Evaluation of the phenolic content and
antioxidant capacities of two Malaysian floral honeys. Food Chemistry, 85 (4),
513–518 .
Baltrušaityte ˙, V., Rimantas-Venskutonis, P., & C ˇeksteryte ˙, V. (2007). Radical
scavenging activity of different floral origin honey and beebread phenolic
extracts. Food Chemistry, 101 (2), 502–514 .
Bogdanov, S. (2002). Harmonized methods of the International Honey
Commission Swiss Bee Research Centre, FAM, Liebefeld, CH 3003 Bern, Switzerland .
Cavia, M. M., Fernández-Muiño, M. A., Huidobro, J. F., Alvarez, C., & Sancho, T.
(2009). Evolution of monosaccharides of honey over 3 years: Influence of
induced granulation. International Journal of Food Science and Technology, 44 (3),
623–628 .
Commision Decision 2002/657/EC of 12 August 2002 (2002). Implementing Council
Directive 96/23/EC concerning the performance of analytical methods and the
interpretation of results. Official Journal of the European Communities, L221 ,
8–36. Brusels, Belgium .
Council Directive 2001/110/EC relating to honey of 20 December 2002, (2002).
Official Journal of the European Communities, L10 , 47. Brusels, Belgium .
Corbella, E., & Cozzolino, D. (2006). Classification of the floral origin of Uruguayan
honeys by chemical and physical characteristics combined with chemometics.LWT Food Science and Technology, 39 (5), 534–539 .
D’Arcy, B. (2005). Antioxidants in Australian floral honeys. Identification of health
enhancing nutrient components. Rural Industries Research and Development
Corporation , 1–84 [Publication no 05/040] .
Escriche, I., Kadar, M., Juan-Borrás, M., & Domenech, E. (2011). Using flavonoids,
phenolic compounds and headspace volatile profile for botanical authentication
of lemon and orange honeys. Food Research International, 44 (5), 1504–1513 .
Escriche, I., Visquert, M., Carot, J. M., Doménech, E., & Fito, P. (2008). Effect of honey
thermal conditions on hydroxymethylfurfural content prior to pasteurization.
Food Science and Technology International, 14 (5), 29–35 .
Escriche, I., Visquert, M., Juan-Borrás, M., & Fito, P. (2009). Influence of simulated
industrial thermal treatments on the volatile fractions of different varieties ofhoney. Food Chemistry, 112 (2), 329–338 .
Escuredo, O., Silva, L. R., Valentao, P., Seijo, M. C., & Andrade, P. B. (2012). Assessing
Rubus honey value: Pollen and phenolic compounds content and antibacterial
capacity. Food Chemistry, 130 (3), 671–678 .
Estevinho, L., Paula Pereira, A., Moreira, L., Dias, L. G., & Pereira, E. (2008).
Antioxidant and antimicrobial effects of phenolic compounds extracts of
Northeast Portugal honey. Food and Chemical Toxicology, 46 (12), 3774–3779 .
Ferreres, F., Andrade, P., & Tomás-Barberán, F. A. (1996). Natural occurrence of
abscisic acid in heather honey and floral nectar. Journal of Agricultural and Food
Chemistry, 44 (8), 2053–2056 .
Ferreres, F., Garcia-Viguera, C., Tomás-Lorente, F., & Tomás-Barberán, F. A. (1993).
Hesperetin: A marker of the floral origin of citrus honey. Journal of the Science of
Food and Agriculture, 61 (1), 121–123 .
Ferreres, F., Giner, J. M., & Tomas-Barberán, F. A. (1994). A comparative study of
hesperetin and methyl antranilate as markers of the floral origin of citrus
honey. Journal of the Science of Food and Agriculture, 65 (3), 371–372 .
Frankel, S., Robinson, G. E., & Berenbaum,, M. R. (1998). Antioxidant capacity and
correlated characteristics of 14 unifloral honeys. Journal of Apicultural Research
& Bee World, 37 (1), 27–31 .
Gheldof, N., Xiao-Hong, W., & Engeseth, N. (2002). Identification and quantification
of antioxidant components of honeys from various floral sources. Journal of
Agricultural and Food Chemistry, 50 (21), 5870–5877 .
Gonzalez-Miret, M. L., Terrab, A., Hernanz, D., Fernandez-Recamales, M. A., &
Heredia, F. J. (2005). Multivariate correlation between color and mineralcomposition of honeys and by their botanical origin. Journal of Agricultural and
Food Chemistry, 53 (7), 2574–2580 .
ICH, Q2B (1996). Validation of analytical procedures: Methodology. In: International
conference on harmonization, Geneva, November, 1–8 .
Iurlina, M., Saiz, A., Fritz, R., & Manrique, G. D. (2009). Major flavonoids of
Argentinean honeys. Optimisation of the extraction method and analysis oftheir content in relationship to the geographical source of honeys. Food
Chemistry, 115 (3), 1141–1149 .
Lachman, J., Hejtmánková, A., Sy ´kora, J., Karban, J., Orsák, M., & Rygerová, B. (2010a).
Content of major phenolic and flavonoid antioxidants in selected Czech honeys.
LWT Czech Journal of Food Sciences, 28 (5), 412–426 .
Lachman, J., Orsák, M., Hejtmánková, A., & Kovárová, E. (2010b). Evaluation of
antioxidant activity and total phenolics of selected Czech honeys. LWT Food
Science and Technology, 43 (1), 52–58 .
Martos, I., Ferreres, F., Yao, L. H., D’Arcy, B. R., Caffin, N., & Tomás-Barberán, F. A.
(2000). Flavonoids in monospecific Eucalyptus honeys from Australia. Journal of
Agricultural and Food Chemistry, 48 (10), 4744–4748 .
Matinez-Vidal, J. L., Aguilera-Luiz, M. M., Romero-Gonzalez, R., & Garrido Frenich, A.
(2009). Multiclass analysis of antibiotic residues in honey by ultraperformance
liquid chromatography–tandem mass spectrometry. Journal of Agricultural and
Food Chemistry, 57 (5), 1760–1767 .
Mato, I., Huidobro, J. F., Simal-Lozano, J., & Sancho, M. T. (2006). Rapid
determination of nonaromatic organic acids in honey by capillary zoneelectrophoresis with direct ultraviolet detection. Journal of Agricultural and
Food Chemistry, 54 (5), 1541–1550 .
Merken, H. M. Y., & Beecher, G. R. (2000). Measurement of food flavonoids by high
performance liquid chromatography: A review. Journal of Agricultural and Food
Chemistry, 48 (3), 577–599 .
Perez, R. A., Iglesias, M. T., Pueyo, E., Gonzalez, M., & De Lorenzo, C. (2007). Amino
Acid composition and antioxidant capacity of Spanish honeys. Journal of
Agriculture and Food Chemistry, 55 (2), 360–365 .
Rand, M., & Bosch-Reig, F. (1998). Classification of Spanish unifloral honeys by
discriminant analysis of electrical conductivity, color, water content, sugars and
pH.Journal of Agricultural and Food Chemistry, 46 (2), 393–400 .
Saxena, S., Gautam, S., & Sharma, A. (2010). Physical, biochemical and
antioxidant properties of some Indian honeys. Journal of Food Chemistry,
118, 391–397 .
Schramm, D. D., Karim, M., Schrader, H. R., Holt, R. R., Cardetti, M., & Keen, C. L.
(2003). Honey with high levels of antioxidants can provide protection to
healthy human subjects. Journal of Agricultural and Food Chemistry, 51 (6),
1732–1735 .
Soler, C., Gil, M., Garcia-Viguera, C., & Tomás-Barberan, F. A. (1995). Flavonoid
patterns of French honeys with different floral origin. Apidologie, 26 (1), 53–60 .
Terrab, A., Gonzalez, G. A., Diez, M. J., & Heredia, F. J. (2003). Mineral content and
electrical conductivity of honeysproduced in Northewet Morocco and their
contribution to the characterisation of unifloral honeys. Journal of the Science of
Food and Agriculture, 83 (7), 637–643 .
Tomás-Barberán, F. A., Martos, I., Ferrerer, F., Radovic, B. S., & Anklam, E. (2001).
HPLC flavonoid profiles as markers for the botanical origin of European unifloral
honeys. Journal of the Science of Food and Agriculture, 81 (5), 485–496 .
Turkmen, N., Sari, F., Poyrazoglu, E. S., & Velioglu, Y. S. (2006). Effects of prolonged
heating on antioxidant activity and colour of honey. Food Chemistry, 95 (4),
653–657 .
Vela, L., De Lorenzo, C., & Perez, R. A. (2007). Antioxidant capacity of Spanish
honeys and its correlation with some physico-chemical parameters and
polyphenolic content. Journal of the Science of Food and Agriculture, 87 (6),
1069–1075 .
Von Der Ohe, W., Persano, L., Piana, M. L., Morlot, M., & Martin, P. (2004).
Harmonized methods of melissopalynology. Apidologie, 35 (1), 18–25 .
Wang, X. H., Gheldof, N., & Engeseth, N. J. (2004). Effect of processing and storage on
antioxidant capacity of honey. Journal of Food Science, 69 (2), 96–101 .
Yao, L. H., Datta, N., Tomás-Barberán, F. A., Ferreres, F., Martos, I., & Singanusong, R.
(2003). Flavonoids, phenolic acids and abscisic acid in Australian and New
Zealand Leptospermum honeys. Food Chemistry, 81 (2), 159–168 .
Yao, L., Jiang, Y., D’arcy, B., Singanusong, R., Datta, N., Caffin, N., et al. (2004).
Quantitative high-performance liquid chromatography analyses of flavonoids in
Australian eucalyptus honeys. Journal of Agricultural and Food Chemistry, 52 (2),
210–214 .I. Escriche et al. / Food Chemistry 142 (2014) 135–143 143