Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis.

Abstract

In this paper is described the applicability of a flow injection system, operating with an amperometric detector, for measurement in rapid and simple way the antioxidant power of honey, propolis and royal jelly. The proposed method evaluates the reducing power of selected antioxidant compounds and does not require the use of free radicals or oxidants. Twelve honey, 12 propolis and 4 royal jelly samples of different botanical and geographical origin were evaluated by the electrochemical method and the data were compared with those obtained by the DPPH assay. Since a good correlation was found (R(2)=0.92) the proposed electrochemical method can be successfully employed for the direct, rapid and simple monitoring of the antioxidant power of honeybee products. Furthermore, the total phenolic content of samples was determined by the Folin-Ciocalteau procedure and the characteristic antioxidant activities showed a good correlation with phenolics (R(2)=0.96 for propolis and 0.90 for honey).

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Talanta 71 (2007) 1387–1392
Evaluation of the antioxidant power of honey, propolis and royal jelly by
amperometric flow injection analysis
S. Buratti∗, S. Benedetti, M.S. Cosio
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universit` a degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy
Received 31 March 2006; received in revised form 15 May 2006; accepted 6 July 2006
Available online 22 August 2006
Abstract
In this paper is described the applicability of a flow injection system, operating with an amperometric detector, for measurement in rapid and
simple way the antioxidant power of honey, propolis and royal jelly. The proposed method evaluates the reducing power of selected antioxidant
compounds and does not require the use of free radicals or oxidants. Twelve honey, 12 propolis and 4 royal jelly samples of different botanical and
geographicaloriginwereevaluatedbytheelectrochemicalmethodandthedatawerecomparedwiththoseobtainedbytheDPPHassay.Sinceagood
correlationwasfound(R2=0.92)theproposedelectrochemicalmethodcanbesuccessfullyemployedforthedirect,rapidandsimplemonitoringof
the antioxidant power of honeybee products. Furthermore, the total phenolic content of samples was determined by the Folin–Ciocalteau procedure
and the characteristic antioxidant activities showed a good correlation with phenolics (R2=0.96 for propolis and 0.90 for honey).
© 2006 Elsevier B.V. All rights reserved.
Keywords: Antioxidant power; Honeybee products; Flavonoids; Amperometric detection; Flow injection system
1. Introduction
In recent years there has been a renewed interest in the study
of honey and propolis composition and biological properties.
Theuseofhoneyandpropolisinthetreatmentandpreventionof
numerous diseases has been documented [1–3]. This beneficial
role is partially attributed to honey and propolis antibacterial
activity. However, since some of these diseases are a conse-
quence of oxidative damage, it seems that part of the therapeu-
tical properties of honeybee products is due to their antioxidant
capacity. Furthermore, honey, as a source of antioxidants, has
been proven to be effective against deteriorative oxidation reac-
tion in food [4,5].
Honeyandpropolistypicallycontainmorethan150polyphe-
nolic compounds, including flavonoids and cinnamic acid
derivatives. It is well known that the chemical properties of
phenolic acids or flavonoids, in terms of the availability of the
phenolic hydrogens as hydrogen donating radical scavengers,
predict their antioxidant properties. Bors [6], by first, proposed
three key determinants in the chemical structure of flavonoids:
∗Corresponding author. Tel.: +39 02 50316623; fax: +39 02 50316632.
E-mail address: susanna.buratti@unimi.it (S. Buratti).
(1) the 3?,4?-dihydroxy catechol configuration on the B ring; (2)
the 2,3-double bond in conjugation with the 4-carbonyl group
on the C ring; (3) the 3-hydroxy group in combination with the
2,3-double bond. More recently other studies about structure
and antioxidant power relationships of flavonoids and pheno-
lic acids were published. In all these works the rule of 3-OH
group attached to the 2,3-double bond and adjacent to the 4-
carbonyl in C ring was confirmed, and also the importance of
theBringhydroxylationontheantioxidantactivityofflavonoids
was demonstrated [7–9].
In literature, several studies for the identification and quan-
tification of antioxidant components of honeybee products are
reported [10,11]. The studies for the evaluation of the antioxi-
dantcapacityofhoneyandpropolisinvolvedtheuseofdifferent
methods such as the ORAC, FRAP and DPPH assays [12,13].
Little information is available on the antioxidant capacity and
chemical composition of royal jelly [14].
The principal aim of our work was to develop a simple and
rapidmethodtoexaminethetotalantioxidantpowerofhoneybee
products.Thismethodisbasedontheelectrochemicalbehaviour
of flavonoids that depends on their chemical structure. In pre-
vious works [15–17] we have demonstrated that the electro-
chemicalbehaviourofphenolicacids,carotensandxanthophylls
can provide a chemical basis for describing their antioxidant
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2006.07.006

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S. Buratti et al. / Talanta 71 (2007) 1387–1392
functionalityandtheresultsobtainedonwine,oliveoilandveg-
etables were perfectly in accordance with those obtained by the
application of the ABTS radical cation decoloration assay [18].
The proposed method evaluates the overall reducing power
ofantioxidantcompoundspresentinsamples.Indespiteofother
assays,itdoesnotinvolvereactivespecies,andcanbeconsidered
a direct test of the total antioxidant power since it is based only
on the chemico-physical properties of antioxidant compounds.
Thisisimportantsincethedependenceonfreeradicaloroxidant
is one of the major drawbacks of currently used methods.
The proposed method was applied to honey, propolis and
royal jelly samples of different botanical and geographical ori-
gins, since these factors greatly influence the chemical com-
position and consequently the antioxidant power of honeybee
products. In particular, unifloral honey samples coming from
different Italian regions, propolis and royal jelly samples of dif-
ferent nations of origin were considered.
In addition, we investigated whether the variation in antiox-
idant content among unifloral honey and propolis samples is
correlated significantly with their phenolic contents.
2. Experimental
2.1. Reagents
Caffeic acid, p-coumaric acid, ferulic acid and DPPH were
purchased from Sigma (St. Louis, MO, USA). Isorhamnetin,
kaempferol, quercetin, galangin, hesperitin, naringenin were
obtained from Extrasynthese (Genay, France); chrysin and
pinocembrin from Funakoshi (Tokyo, Japan). All other chemi-
cals were supplied by Sigma.
2.2. Samples
Twelve different Italian unifloral honey samples, four royal
jelly samples and six crude propolis samples of specific geo-
graphical origins were analysed. All these samples were pro-
duced in the same year and were provided by the Istituto
Nazionale di Apicoltura (Bologna, Italy). The honey unifloral
authenticitywasverifiedintheaccreditedlaboratoryofthisinsti-
tutebypollen,organolepticandchemicalanalyses.Datawerein
accordancewiththeunifloralhoneycharacterizationestablished
bytheItalianMinisterofAgriculture[19].Othersixcommercial
hydroalcoholic propolis preparations were obtained from local
markets. The year of production and the origin of the commer-
cial propolis samples were not known. Samples, once received,
were stored at 4◦C until analysis. In Table 1 the list of samples
and their geographical and/or botanical origins is reported.
2.3. Sample preparation
Before injection in the Flow Injection (FI) system the honey-
bee products were diluted with methanol as described below:
- honey samples 1:10 (w/v);
- royal jelly samples 1:20 (w/v);
- commercial propolis samples 1:50 (w/v).
Table 1
List of samples and their geographical and/or botanical origins
Geographical originBotanical origin
Honey
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
Sicilia
Sicilia
Campania
Sardegna
Lombardia
Trentino
Piemonte
Val d’aosta
Toscana
Lombardia
Lombardia
Piemonte
Citrus spp.
Citrus spp.
Citrus spp.
Citrus spp.
Rhododendron spp.
Rhododendron spp.
Rhododendron spp.
Rhododendron spp.
Rhododendron spp.
Robinia pseudoacacia L.
Robinia pseudoacacia L.
Robinia pseudoacacia L.
Propolis
P1
P2
P3
P4
P5
P6
P7a
P8a
P9a
P10a
P11a
P12a
Italy
Germany
Italy
Hungary
China
China
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Royal jelly
RJ1
RJ2
RJ3
RJ4
China
China
Italy
Italy
–
–
–
–
aCommercial hydroalcoholic propolis extracts.
After dilution samples were sonicated and centrifugated at
10,000×g for 10min. One gram of crude propolis samples was
extractedwithethanolatroomtemperaturefor24h.Theethanol
solution was filtered through a 0.45?m filter (Whatman, Maid-
stone,England)anddiluted1:50withmethanolbeforeinjection.
2.4. Electrochemical method
The flow injection apparatus consisted of a Jasco (Tokyo,
Japan) model 880 PU pump an EG&G Princeton Applied
Research (Princeton, NJ) model thin-layer electrochemical
detector equipped with a single glassy carbon electrode oper-
ating at a potential of +0.5V, a reference electrode (Ag/AgCl
saturated), and a platinum counter electrode. The connecting
tubes were of PEEK (1.5mm o.d.×0.5mm i.d). Data were
recorded using a Philips (Eindhover, Netherlands) PM 8252
recorder. Analyses were performed at room temperature using
a carrier solution composed of methanol 700mll−1, sodium
acetate–acetic acid buffer (0.1M, pH 4.5) 300mll−1, sodium
perchlorate monohydrate 20gl−1. A flow rate of 0.7mlmin−1
was employed. The FI experiments were performed operating
in amperometric mode and the antioxidant power of honeybee
products was evaluated by measuring the current resulted from

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S. Buratti et al. / Talanta 71 (2007) 1387–1392
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Fig. 1. Chemical structures of flavonoids and cinnamic acids analysed.
theoxidationofelectroactivecompoundspresentinthesamples,
attheestablishedpotentialof+0.5V(versusAg/AgCl).Further-
more, hydrodynamic voltammetric experiments were carried
out in order to investigate the electrochemical behaviour of
the main phenolic compounds identified in honeybee products.
The hydrodynamic voltammograms of these compounds were
obtained by running a series of FI experiments in which the
potential was increased in the range between +0.2V and +0.8V
(versusAg/AgCl)bystepof0.1Vandthecurrentwasmeasured.
2.5. DPPH radical scavenging activity
The antioxidant activity of honey, propolis and royal jelly
samples was also evaluated using the DPPH (2,2-diphenyl-1-
picrylhydrazyl free radical) assay according to the method of
Brand-Williams [20]. Different dilutions of honeybee samples
dissolved in methanol were added to 3.9ml of a 6.1×10−5M
DPPH•methanolicsolution.TheexactinitialDPPH•concentra-
tion (CDPPH•) in the reaction medium was calculated by a cali-
bration curve (Abs515=11771×(CDPPH•)+0.01). The bleach-
ing of DPPH•was monitored at 515nm (Spectrophotometer
Uvidec-610,Jasco,Tokyo,Japan)for30minagainstablankcon-
stituted by the honeybee solution containing all reagents except
DPPH•. The scavenging activity of the samples on the DPPH•
was expressed as IC50(mgml−1) and was extrapolated from a
dose–response curve. All analyses were carried out in triplicate.
2.6. Determination of total phenolics in honey and propolis
Honey and propolis samples were extracted with ethyl
acetate, as described by Floris [21]. The total phenolic con-
tents were determined by the Folin–Ciocalteau procedure [22].
Absorbance was read at 760nm, and phenols were deter-
mined using a calibration curve for 0.5mgkg−1, 1mgkg−1,
2.5mgkg−1, 5mgkg−1, 10mgkg−1of caffeic acid.
3. Results and discussion
A preliminary study on the electrochemical behaviour of
flavonoids and phenolic acids typically present in honeybee
products was performed in order to establish the best electro-
chemical conditions for the evaluation of the total antioxidant
power of propolis, honey and royal jelly.
In Fig. 1 the chemical structures of the assayed compounds
are reported.
The hydrodynamic voltammetric profile of these compounds
(Fig. 2) shows their oxidative behaviour in relation to the
established potential. Since the oxidation potential provides
an estimate of the energy required to donate an electron, the
lowest the oxidation potential the most easily the compound
will donate an electron and the highest will be its expected
antioxidant power. As it is possible to see at potential lower
than +0.5V (versus Ag/AgCl), only the flavonols and the cin-

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S. Buratti et al. / Talanta 71 (2007) 1387–1392
Fig. 2. Hydrodynamic voltammetric profiles of flavonoids and cinnamic acids.
Operative conditions: standard concentrations, 3.7×10−5moll−1; flow rate,
0.6mlmin−1; carrier solution—methanol, 700mll−1; sodium acetate–acetic
acidbuffer,300mll−1;sodiumperchloratemonohydrate,20gl−1;injectionvol-
ume, 20?l.
Fig. 3. Linear correlation plot of the DPPH assay (1/IC50) and the electrochem-
ical method.
namic acid derivatives can be detected while flavones and fla-
vanons are oxidizable only at potential greater than +0.6V (ver-
sus Ag/AgCl). The potential of +0.5V appears to be selective
to discriminate only the compounds having effective antioxi-
dant power and at this potential the reducing power decreases
in order: quercetin>isorhamnetin>kampferol>caffeic acid>
Table 2
Antioxidant power and total phenols content of honeybee samples (average±s) (n=3)
Electrochemical method (mgg−1galangin)DPPH method IC50(mgml−1)Total phenols (mg100g−1caffeic acid)
Honey
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
17 ± 1
27 ± 1
43 ± 3
19 ± 1
34 ± 2
24 ± 4
42 ± 3
32 ± 2
14 ± 1
23 ± 3
19 ± 1
22 ± 1
15.1 ± 0.4
6.9 ± 0.4
5.0 ± 0.3
11.0 ± 0.4
6.1 ± 0.5
7.1 ± 0.5
5.7 ± 0.3
6.2 ± 0.3
15.5 ± 0.8
8 ± 1
12.0 ± 0.6
8 ± 1
20.4±0.5
29.2±0.9
60±1
24.7±0.9
38±2
31.2±0.9
56±1
37±1
17.1±0.7
36.8±0.7
20.9±0.4
30.8±0.9
Propolis
P1
P2
P3
P4
P5
P6
P7a
P8a
P9a
P10a
P11a
P12a
167 ± 5
103 ± 3
99 ± 2
115 ± 3
94 ± 2
27 ± 0.3
129 ± 3
35 ± 1
2 ± 0.1
26 ± 0.4
6 ± 0.3
5 ± 0.3
1.0 ± 0.1
1.5 ± 0.1
2.1 ± 0.2
1.8 ± 0.1
2.2 ± 0.1
6.6 ± 0.5
1.9 ± 0.1
3.4 ± 0.2
36 ± 1
4.4 ± 0.4
6.8 ± 0.1
79 ± 2
181±4
105±3
108±3
114±3
99±3
29.0±0.6
170±3
55.0±0.6
2.0±0.1
45±1
7.6±0.1
7.3±0.1
Royal jelly
RJ1
RJ2
RJ3
RJ4
32 ± 1
27 ± 1
110 ± 3
96 ± 2
3.7 ± 0.2
7.0 ± 0.5
1.4 ± 0.1
2.3 ± 0.2
–
–
–
–
aCommercial hydroalcoholic propolis extract.

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Fig. 4. Linear correlation plots of antioxidant power of propolis and honey vs. total phenols.
galangin>ferulicacid>p-cumaricacid.Hesperetin,naringenin,
chrysin and pinocembrin are not revealed and presumably they
haveverylittleornoantioxidanteffect.Asitispossibletonotice
flavonols show a reducing power higher than flavons and fla-
vanons.Theseresultsareinaccordancewithotherworks[23,24]
and are explained by their chemical structures.
Among the cinnamic acid derivatives caffeic acid shows an
antioxidantpowerstrongerthanferulicacidandp-cumaricacid.
The same result has been obtained in other works in which the
hydrocinnamateswerescreenedfortheirradicalscavengeractiv-
ity [25].
On the basis of these preliminary results, a FI system with
electrochemical detector operating at a potential of +0.5V (ver-
sus Ag/AgCl) was used for the determination of the antioxidant
power of honeybee products.
The antioxidant power was quantified using galangin as ref-
erence. Quantification was based on peak height. The amper-
ometric detection resulted in well-defined linear concentration
dependenceinarangebetween1mgl−1and10mgl−1ofgalan-
gin. The relative standard deviation at the concentration level of
5mgl−1was2.5%(n=15)andtheestimateddetectionlimit,cal-
culatedinaccordancetoMillerandMiller[26],was0.15mgl−1.
Several samples of honey, propolis and royal jelly were anal-
ysedbytheproposedprocedureandtheresultswereexpressedas
mgg−1galangin equivalent. The same samples were also anal-
ysed by the DPPH assay and their total phenolic content was
determined by the Folin–Ciocalteau procedure.
In Table 2 the total phenolic content and the antioxidant
power of the honeybee products evaluated by the electrochem-
ical method and by the DPPH assay are reported. The data
obtained by the proposed method were substantially confirmed
by the DPPH assay since a good correlation (R2=0.92) was
observed between the two methods (Fig. 3).
Consideringthedataobtainedbytheelectrochemicalmethod
it can be notice that there is a great variation in the antioxi-
dant power among the investigated honeybee products. In par-
ticular the antioxidant power of propolis samples varies from
2mgg−1to 169mgg−1galangin equivalent, crude propolis
samples exhibit an antioxidant power higher than commercial
hydroalcoholic propolis preparations except for sample P7. The
twoChinesepropolishaveanantioxidantpowerlowerthanItal-
ian and German samples.
The antioxidant power of honey samples varies from
14mgg−1to 43mgg−1. Except for a Citrus sample (H3),
Rhododendronsamples(H5–H8)exhibitthehighestantioxidant
power while Robinia (H9–H12) the lowest. The variation in the
antioxidant power among unifloral honeys with different geo-
graphicalorigincanbeduetoclimateandenvironmentalfactors
such as humidity, temperature and soil composition.
The antioxidant power of the two Italian royal jelly appears
to be higher than the two Chinese samples.
Total phenolic content ranges from 2.0mgg−1to 181.0mg
g−1caffeic acid equivalent for propolis, and from 17.1mgg−1
to 60.3mgg−1for honey.
Regressionanalysisbetweenantioxidantpowerandtotalphe-
nols content shows a linear correlation (Fig. 4). Coefficients of
R2=0.96 and 0.90 have been obtained for propolis and honey,
respectively. These results suggest that the antioxidant power of
propolis and honey samples is especially due to phenolic com-
pounds that are the major constituents having reducing power.
The antioxidant power of honey probably can be related also
to other reducing agents present at low concentration and not
revealed by the Folin–Ciocalteau procedure.
4. Conclusion
The results suggest that the electrochemical method can
be successfully used for monitoring the antioxidant power of
honeybee products. With respect to the other reported meth-
ods, the proposed offers significance improvement since it is
rapid (60determinationsh−1), simple and does not require the
use of free radicals or oxidants since it evaluates the overall
reducing power of antioxidant compounds present in the sam-
ple. Furthermore, the data collected demonstrate that within the
honeybee products the crude propolis has the highest antioxi-
dant power followed by royal jelly and honey. Among honey
samples, Rhododendron exhibits the highest antioxidant power;
among commercial propolis there is a great variation in the