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Antioxidant and Radical Scavenging Activity of Honey in Endothelial Cell Cultures (EA.hy926)


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The therapeutic properties of honey, once considered a form of folk or preventive medicine, are acquiring importance for the treatment of acute and chronic free radical-mediated diseases (atherosclerosis, diabetes and cancer). The aim of this work was to study the protective activity of a honey of multifloral origin, standardized for total antioxidant power and analytically profiled (HPLC-MS) in antioxidants, in a cultured endothelial cell line (EA.hy926) subjected to oxidative stress. Cumene hydroperoxide (CuOOH) was used as free radical promoter. Native honey (1% w/v pH 7.4, 10(6) cells) showed strong quenching activity against lipophilic cumoxyl and cumoperoxyl radicals, with significant suppression/prevention of cell damage, complete inhibition of cell membrane oxidation, of intracellular ROS production and recovery of intracellular GSH. Experiments with endothelial cells fortified with the isolated fraction from native honey enriched in antioxidants, exposed to peroxyl radicals from 1,1-diphenyl-2-picrylhydrazyl (AAPH, 10 mM) and to hydrogen peroxide (H2O2, 50-100 microM), indicated that phenolic acids and flavonoids were the main causes of the protective effect. These results provide unequivocal evidence that, through the synergistic action of its antioxidants, honey by reducing and removing ROS, may lower the risks and effects of acute and chronic free radical induced pathologies in vivo.
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There is an increasing need for a better definition of the thera-
peutic properties of honey, which are restricted to its dermatolo-
gical beneficial effects: honey reduces skin inflammation, edema
and exudation, promotes wound healing, diminishes scar size
and stimulates tissue regeneration [1]. However, it may also
give benefit in more severe pathologies, i.e., gastrointestinal ul-
cerative diseases, atherosclerosis and cancer where free radicals
play a key role [2]. Hamzaoglu reported that tumor implantation
in rats was markedly reduced by the application of honey pre-
and post-operatively, suggesting that the physico-chemical ac-
tion (decrease of oxygen availability in the tumor environment,
i.e., anti-angiogenic effect) and its antioxidants can prevent the
spread of metastatic cells [3]. Honey products (given per os)
have also proved successful in the prevention and therapy of
murine transplantable tumors [4], [5].
Until 1990, the chemopreventive action of honey was attributed
to its hydrogen peroxide-releasing properties, through induction
of cell apoptosis [6], [7], but recent findings point to a comple-
mentary role of the phytochemical antioxidants which can act
synergistically or independently from the release of H
Honey contains an array of chemicals endowed with antiradi-
Antioxidant and Radical Scavenging Activity of Honey
in Endothelial Cell Cultures (EA.hy926)
Giangiacomo Beretta
Marica Orioli
Roberto Maffei Facino
Istituto di Chimica Farmaceutica e Tossicologica ªPietro Pratesiº, Faculty of Pharmacy, University of Milan,
Milan, Italy
Dr. Giangiacomo Beretta ´ Istituto di Chimica Farmaceutica e Tossicologica ´ Faculty of Pharmacy ´ University of
Milan ´ viale Abruzzi 42 ´ 20131 Milan ´ Italy ´ Phone: +39-02-5031-7519 ´ Fax: +39-02-5031-7565 ´
Received April 6, 2007 ´ Revised July 9, 2007 ´ Accepted July 17, 2007
Planta Med 2007; 73: 1182±1189 Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2007-981598 ´ Published online September 7, 2007
ISSN 0032-0943
The therapeutic properties of honey, once considered a form of
folk or preventive medicine, are acquiring importance for the
treatment of acute and chronic free radical-mediated diseases
(atherosclerosis, diabetes and cancer). The aim of this work was
to study the protective activity of a honey of multifloral origin,
standardized for total antioxidant power and analytically pro-
filed (HPLC-MS) in antioxidants, in a cultured endothelial cell
line (EA.hy926) subjected to oxidative stress. Cumene hydroper-
oxide (CuOOH) was used as free radical promoter. Native honey
(1% w/v pH 7.4, 10
cells) showed strong quenching activity
against lipophilic cumoxyl and cumoperoxyl radicals, with sig-
nificant suppression/prevention of cell damage, complete inhibi-
tion of cell membrane oxidation, of intracellular ROS production
and recovery of intracellular GSH. Experiments with endothelial
cells fortified with the isolated fraction from native honey enrich-
ed in antioxidants, exposed to peroxyl radicals from 1,1-diphe-
nyl-2-picrylhydrazyl (AAPH, 10 mM) and to hydrogen peroxide
M), indicated that phenolic acids and flavonoids
were the main causes of the protective effect. These results pro-
vide unequivocal evidence that, through the synergistic action of
its antioxidants, honey by reducing and removing ROS, may low-
er the risks and effects of acute and chronic free radical induced
pathologies in vivo.
Key words
Honey ´ endothelial cells ´ radical scavenging activity ´ antioxi-
dant fraction ´ GSH
Original Paper
cal/anti-inflammatory activity, i.e., phenolic derivatives which
can play roles, alone or in combination, in its anti-tumor, anti-in-
flammatory effects [9]. The anti-tumoral effect of honey seems to
be due to a multifactorial process: 1) release of cytotoxic H
(and of HO radicals after Fenton reaction) [7]; 2) a direct inhibi-
tion of COX-2 by some specific constituent (chrysin and caffeic
acid phenyl ethyl ester, CAPE) [10]; and 3) scavenging action
against different reactive oxygen species (ROS) responsible for
induction of the inflammatory burst, which if not properly quench-
ed/contained can degenerate into cell malignancy [11]. Several
studies have focused on the antioxidant power of different types
of honey, primarily seeking to identify its floral and geographical
origin [12], [13], [14] and none of them have investigated the
protective effect of this complex matrix in living cells subjected
to oxidative stress. This was the aim of the present work.
We used: 1) a well established human cell culture line (endothe-
lial cells, EA.hy926); 2) a set of different free radical promoters
, AAPH); 3) a prototype honey of multifloral origin,
standardized in antioxidant activity according to different meth-
ods [Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), fer-
ric reducing antioxidant power (FRAP), and oxygen radical absor-
bance capacity, (ORAC)] [15]; and 4) an isolated fraction from
multiflora honey uniformly enriched in antioxidant constituents,
whose structure was analytically profiled by conventional tech-
niques (LC-UV/DAD-MS). Our aim was to find out whether the
cytoprotective activity of honey arose from a free radical scaven-
ging action of these constituents, and provide a mechanistic ex-
planation of the results.
Materials and Methods
Chemicals and cell lines
The organic solvents were all of analytical grade (Sigma-Aldrich;
Milan, Italy). 2,2
-Azobis(2-amidinopropane) dihydrochloride
(AAPH) was from Wako Chemicals (Società Italiana Chimici;
Rome, Italy),
-phycoerythrin (
-PE) from Porphydium cruentum,
6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), and N-
acetylcysteine (NAC) were purchased from Sigma±Aldrich. 1,1-Di-
phenyl-2-picrylhydrazyl (DPPH), cumene hydroperoxide (CuOOH),
Folin-Ciocalteu reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), o-phenyl-aldehyde (OPA) FeCl
O and hydrogen peroxide 30% were from Fluka
(Buchs, Switzerland). 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-
bora-3a,4a-diaza-S-indacene-3-undecanoyl acid (C
-BODIPY 581/
591, BODIPY) and 2
-dichlorodihydrofluorescein diacetate
(DCFH-DA) were from Molecular Probes (Space Import±Export;
Milan, Italy). Chrysin, galangin, pinocembrin, luteolin, caffeic
acid, cinnamic acid, p-coumaric acid, syringic acid, benzoic acid,
Amberlite XAD-2, Dulbecco's phosphate buffered saline (PBS),
Dulbecco's modified Eagle's medium (DMEM), penicillin and
streptomycin, HAT media supplement, and
L-glutamine were all
from Sigma. Human endothelial cells (EA.hy926) were from Flow
Laboratories (Irvine, UK).
Spectrophotometric apparatus
Spectrophotometric measurements were conducted with a com-
puter-aided Perkin-Elmer UV-VIS spectrophotometer Lambda 16
(Perkin-Elmer; Monza, Italy). Fluorimetric determinations were
carried out with a Wallac Victor
Multiwell instrument (Perkin-
Elmer) with fluorescent filters (
= 485 nm;
= 535 nm).
Commercial multiflora honey was purchased in a supermarket
(Milan, Italy), stored at 4 8C in the dark until tested by the usual
physicochemical tests (pH, electrical conductivity, titrable acid-
ity, ash content) and by qualitative tests [Lugol test and diastases
index for authenticity, hydroxymethylfurfural test (HMF) for
quality] and quantitative tests (reducing and non-reducing su-
gars) [16]. A sugar analogue consisting of 40% fructose, 30% glu-
cose, 10% maltose and 20 % water was used to check whether the
main sugar components of honey interfere in the assays.
Total phenolics and antioxidant activity in honey
The total phenol content of whole honey was determined by a
modification of the Folin-Ciocalteu method, and Trolox
used as standard to plot the calibration curve. The radical
scavenging activity of each XAD-2 honey fraction was evaluated
using different approaches (FRAP, DPPH and ORAC) according to
the methods previously described by us [15]. ORAC values are
expressed as Trolox equivalent (TE).
Cell cultures
Endothelial cells were cultured in DMEM supplemented with
10 % fetal calf serum, 2 mM
L-glutamine, antibiotics (100 U/mL
penicillin and 0.1 mg/mL streptomycin) and HAT supplement.
The cells were maintained in a humidified 5% CO
and sub-cultured 1:3 twice a week. All experiments were con-
ducted at 378C.
Model 1: protective effect of honey against intra-cellular and
cell membrane lipid oxidation
The cell monolayer was washed twice with the pre-warmed PBS
(pH 7.4) and then exposed to CuOOH (100
M) for 10 minutes.
The cells were then washed twice to remove the oxidant and
two types of fluorescent probe were incorporated: 1) BODIPY to
evaluate cell membrane oxidation, and 2) DCFH-DA for the intra-
cellular oxidative damage. Both the probes were incubated at 10
M (PBS) for 30 min. Then, the cell cultures were washed twice
with PBS and incubated with the following solutions:
± PBS (control)
± Honey solution (1% w/v in PBS)
± Trolox solution (30
M in PBS)
± Honey blank (1% w/v sugar analogue in PBS)
The membrane and intracellular oxidation kinetics were moni-
tored with a Victor
Wallac multi-well fluorescent plate reader
(excitation filter 500 10 nm; emission filter 530 12.5 nm)
using the following instrumental parameters: CW lamp energy
15,000 (arbitrary units), irradiation time 0.1 s, and cycle time 5
min for 46 cycles (time analysis = 60 min).
DCFH-DA assay
The DCFH-DA stock solution was prepared in ethanol (3.3 mM)
and stored under nitrogen at ±208C. Intracellular oxidative dam-
age was investigated by the DCFH-DA assay [17]. The endothelial
cells were washed twice with the pre-warmed PBS, incubated
with a DCFH-DA solution in PBS (10
M for 10 min), and the
probe incorporation was confirmed by fluorescence microscopy
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ±1189
Original Paper
(20 Ph1 ADL lens and a FITC filter -Eclipse TS 100; Nikon; Tokyo,
The cell monolayer was washed twice with PBS, then incubated
with the following solutions:
± Honey blank
± Honey solution (1% w/v in PBS)
± Trolox solution (30
M in PBS)
The fluorescence intensity of DCF, the DCFH-DA oxidation prod-
uct, was monitored as described at an excitation wavelength of
485 10 nm and an emission wavelength of 530 12.5 nm for 1
Model 2: preventive effect of honey against acute oxidative
The endothelial cell monolayer was washed twice (PBS), then in-
cubated for 15 h with 1) D-MEM alone; 2) D-MEM supplemented
with 1 % honey (w/vol); 3) 50
M vitamin E, 4) 200
with the honey blank (1% w/vol).
At the end of the pre-treatment, the cells were washed free of the
cell culture medium, the probe BODIPY and the CuOOH incorpo-
rated and the lipid oxidation kinetics were monitored as above
Cell viability
Cell viability was determined by the MTT test as already reported
GSH analysis
Endothelial intracellular GSH was determined as already report-
ed [19].
Preparation of the antioxidant-enriched fraction from honey
Fifty g of the prototype multiflora honey were diluted with four
volumes of distilled water, stirred until completely dissolved, fil-
tered on cotton to remove solid particles and the pH adjusted to
2.5 with 6 N HCl. The filtrate was passed through a column
(25 cm2.5 cm) of Amberlite XAD-2 resin and after sequential
washing first with acidic (200 mL, pH 3) and then with neutral
water (200 mL) [20], the antioxidant-enriched fraction was re-
covered by elution with methanol (200 mL). The methanol frac-
tion was dried under vacuum at 40 8C, taken up in methanol/H
2:1 (4 mg/mL) and analyzed by HPLC-UV-DAD-MS analysis.
All the fractions were tested for the total phenolic content (Folin-
Ciocalteu) and for radical scavenging/antioxidant activity (FRAP,
DPPH etc).
Characterization of honey antioxidant constituents (LC-DAD
and APCI-MS analyses)
HPLC-DAD experiments were carried out on a Thermoquest Sur-
veyor System (Thermoquest; Milan, Italy), equipped with a qua-
ternary pump, a Surveyor Model 6000 LP UV/VIS diode array pro-
grammable detector, operating at 280 nm, a Surveyor AS auto-
sampler, a vacuum degasser and a Xcalibur Software. Compo-
nents were separated with a Phenomenex Synergy RP8 column
(150 mm2 mm i.d.; particle size 4
m) protected with a Max-
RP guard column (4 mm 2 mm i. d.; particle size 4
m). Gradient
elution: 100% solvent A [95% ammonium formate (3.2 mM)/5%
CN, pH 4.5 with HCOOH ] to 40% B [95% CH
CN, 5% ammo-
nium formate (3.2 mM), pH 4.5 with HCOOH] in 30 minutes,
then to 100% solvent B after 10 minutes; flow rate 0.2 mL/min.
APCI-HPLC-MS analysis was conducted with a Thermo Finnigan
LCQ Advantage ion trap mass spectrometer (Thermoquest). Va-
porizer temperature 4508C, ionization voltage 8 kV. Nebulizer
gas (N
) flow rate 0.5 L/min. Spectra were detected in positive
and negative ion mode (100±600 m/z, 0.5 scan/sec).
Cytoprotective activity of honey antioxidant-enriched
The cell monolayer was washed twice with pre-warmed PBS,
then incubated with: 1) BODIPY for cell oxidation, or 2) DCFH-
DA for intracellular oxidative damage. The cells were subjected
to oxidative stress by AAPH (10 mM in PBS), with or without the
honey antioxidant fraction and the oxidative damage was
checked after 1, 2, 3 h incubation times. The H
stress was de-
livered to cell cultures using increasing concentrations of the hy-
droperoxide (50± 100
M in PBS). After 1 h, the oxidative treat-
ment was stopped by washing the monolayers twice with PBS
and the increase of fluorescence of oxidized DCFH-DA was mon-
itored. In parallel cell morphology was evaluated. In both the oxi-
dative assays the antioxidant enriched fractions was incorpora-
ted in the endothelial cells (10 min, 378C) and these exposed to
the free radical inducers.
Statistical analysis
Statistical analyses were conducted with the Prism software for
Windows software package (GraphPad Inc.; San Diego, CA, USA).
Results are expressed as the mean (S.D). of four independent ex-
periments. Student's t-test was used; P values < 0.05 were con-
sidered to be significant.
Results and Discussion
Fig.1 reports the results relative to the cytoprotective activity of
honey and Trolox
in parallel with a honey blank in cell cultures
exposed to oxidative stress by the free radical promoter CuOOH
When endothelial cells were incubated with 100
even for a short time (10 min), there were marked alterations
to the original shape of the cell, as shown by phase-contrast mi-
croscopy (Fig.1A), which were paralleled by a significant loss of
cell viability: 733%vs1003%incontrolcells(p<0.001,
The dramatic change in morphology was caused by membrane
lipid oxidation (see the appearance and time-dependent in-
crease in BODIPY fluorescence, Fig.1C) due to Fe(II)-trace-cata-
lyzed decomposition of CuOOH to cumoxyl and cumoperoxyl
radicals, which diffuse from the membrane lipid bilayer into
the intracellular compartment. In parallel the cell begins to
show preliminary signs of a shrunken cytoplasm, a more rounded
shape, cell surface blebbing, and loss of inter-cellular adher-
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ± 1189
Original Paper
The addition of honey (1% w/v, equivalent to 0.85
reverted the progression of the damage: the BODIPY fluores-
cence, after a slight increase, remained almost constant within
the 1 h oxidation time. Similar inhibition, although less pro-
nounced, was observed for intracellular oxidation: after 1 h the
damage was 60 % less than in unprotected cells (Fig.1D). In the
presence of honey, cell viability slightly but significantly in-
creased, from 73 3% in unprotected cells to 87 4 % in cells
treated with honey (P < 0.05). Hence the antioxidant constitu-
ents of honey can inhibit the free radical species (i. e., cumoxyl
and cumoperoxyl radicals) both at membrane level and intracel-
lularly, limiting the propagation of the oxidative cascade. Alter-
natively these constituents may inactivate cytotoxic CuOOH by
metabolic reduction, through activation of GSH-peroxidase
(GSPx) [22]. In accordance with previous demonstrations in
homogeneous phase models [15], the honey blank did not give
any protective effect.
Then we evaluated the susceptibility of endothelial cells, incuba-
ted overnight with native honey, to CuOOH, checking membrane
lipid peroxidation, cell morphology and viability and intracellu-
lar redox status (GSH).
The cells showed 1) markedly less susceptibility to lipid peroxi-
dation (30 % lower than control cells, Fig. 2A); 2) more limited
morphological alterations (Fig. 2B); 3) a slight increase in cell
viability (15% gain, Fig. 2C).
The basal content of GSH in control cells shown in Fig. 3 (30.0
2.0 nmol/mg protein, column a), was not affected by incubation
with sugar blank (column b), honey (column c) or vitamin E (col-
umn d), but NAC induced a significant rise in intracellular GSH
(42% gain, column e).
Exposure to CuOOH, significantly reduced the level of GSH to 17.9
0.6 nmol/mg protein (column f). The GSH content (28.2 0.2
nmol/mg protein, column j) of cells incubated with NAC and ex-
posed to CuOOH was close to the control value, the smaller drop
being due to the additional GSH pool biosynthesized from the
precursor during the incubation. Most importantly, in cells
grown in DMEM enriched with honey (column h) then challeng-
ed with CuOOH, there was significant sparing of native GSH in
respect to control cells (GSH content 22.4 0.5 nmol/mg vs.
17.9 0.6 nmol/mg, column g, P < 0.05). Vitamin E, deeply and
firmly incorporated within the cell membrane, had no effect
(column i).
It therefore appears that some honey components, which are li-
pophilic, can cross the endothelial cell membrane and 1) spare
the endogenous antioxidant GSH from the oxidative attack of li-
Fig. 1 Protective effect in endothelial
cells (EA.hy926) of 1 % native honey in
PBS against oxidative stress. Free radical
promoter: CuOOH (100
M); control
(CTR): sugar blank (1 % w/v, PBS); Tro-
M). A Phase contrast micro-
scopy. B Cell viability (MTT test). C
Membrane lipid peroxidation (BODIPY).
D Intracellular ROS production (DCFH).
Results are expressed as the mean
S.D. of 4 independent experiments. * P
< 0.05, Student's t test, vs. CuooH + su-
gar blank.
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ± 1189
Original Paper
Fig. 2 Preventive effect of native hon-
ey (1 % PBS) against CuOOH (100
promoted oxidative stress. Control
(CTR): sugar blank (1 % w/v, PBS). A
Membrane lipid peroxidation (BOPIDA).
B Phase contrast microscopy. C Cell via-
bility (MTT test). Results are expressed
as the mean S.D. of 4 independent ex-
Fig. 3 Intracellular GSH levels in endothelial cells pre-
treated overnight with: 1 % sugar blank, 1 % native honey,
M vitamin E and 200
M N-acetylcysteine (NAC). Unex-
posed cells (columns a ± e) and 100
M CuOOH exposed
cells (columns f ± l). Results are exprerssed as te mean
S.D. of 4 independent ewxperiments. * P < 0.05 Student's t
test, column h vs g.
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ± 1189
Original Paper
pophilic peroxyl radicals, and/or 2) regenerate GSH from GSSG by
a hydrogen-transfer mechanism, or both.
Finally, we evaluated the protective activity of the antioxidant
fraction isolated from native honey. Cells were exposed to a flux
of peroxyl-radicals generated by AAPH (10 mM) or to H
and 100
M). Fig. 4 shows the HPLC profile of the antioxidant
fraction of multiflora honey, containing several constituents (Ta-
ble 1). These were identified on the basis of the UV-DAD profile,
MS fragmentation pattern, and by comparison of the chromato-
graphic, spectroscopic and MS properties of reference standards
(not shown), as caffeic acid, p-coumaric acid, eriodictiol, pino-
banksin, chrysin, pinocembrin, and galangin (typical of propolis
and beeswax), and as kaempferol and apigenin (from pollen).
There was a marked increase in membrane lipid-peroxidation in
unprotected endothelial cells exposed to AAPH (see the time-de-
pendent increase of BODIPY fluorescence, Fig. 5A), with no signs
of morphological alterations. Addition of the antioxidant fraction
(5, 10, 15
cells), induced a dose- and time-dependent
decrease in the membrane lipid peroxidation, which was sup-
pressed at 15
cells. Comparing this protective effect
with that given by 1% native honey (0.85
cells), the
latter is from 15 to 20-fold more active than its antioxidant frac-
tion. This suggests that by the chromatographic sorbtion method
adopted [12], [13], [14], [21], [23], we lose an appreciable amount
of antioxidant in the filtrate and in the first washings which can
synergistically concur with those eluted with methanol in the
cytoprotective activity of multiflora honey. Our assays of total
phenol content and total antioxidant activity gave direct evi-
dence for an overall loss in the washings of antioxidant material
of approximately 40% in respect to honey as such (not shown).
Very likely highly polar, hydrophilic antioxidant phenolics elute
in the first washings embedded in sugars. This is in accordance
with the data of Gheldof et al. and Weston et al. [21], [24], who
using the same method, found more than 40% of the total anti-
bacterial and antioxidant activity of honey in the first washings.
This phenol-positive material is not extractable with organic sol-
vents (unpublished data). However, it is also possible that some
other antioxidants in the matrix, structurally unrelated to poly-
phenols (vitamin E, carotenoids, small proteins, amino acids),
may not be detected by our LC-MS method. These too can play a
role in the cytoprotective activity of multiflora honey. Overall,
these considerations help to explain the contradictory reports
Table 1 Main components of the antioxidant enriched fraction from multiflora honey (RT retention time, MW molecular weight)
Peak number RT (min) MW (a. m. u.) Compound
1 6.43 467 convicine diglucoside*
2 10.50 163 4-N-aziridylbenzoic acid*
3 10.55 165 4-dimethylaminobenzoic acid*
4 17.92 180 caffeic acid
5 26.23 164 p-coumaric acid
6 39.55 288 eriodictiol
7 43.23 286 kaempferol
8 44.72 270 apigenin
9 45.14 272 pinobanksin
10 46.04 330 quercetin dimethyl ether
11 47.36 330 quercetin dimethyl ether (isomer)
12 48.03 254 chrysin
13 48.35 256 pinocembrin
14 48.52 270 galangin
* Nitrogen-containing compounds 1, 2 and 3 were only tentatively identified.
Fig. 4 HPLC-UV (280 nm) profile of the antioxidant enrich-
ed fraction from native multiflora honey. For structure as-
signments, see Table 1.
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ± 1189
Original Paper
on the biological activity of purified honey fractions compared to
native honey [24], and point to the need for studies and clinical
trials using native honey as such.
When we used the physiological oxidant H
, source of super-
oxide anion by Haber-Weiss reaction or of HO
radical by Fenton
reaction, free to permeate the cell compartments, there was a
strong increase of intracellular ROS (Fig. 5B), due to depletion of
GSH. Because of the weak oxidative conditions, close to in vivo
situations, phase contrast microscopy did not show up any mor-
phological alterations (not shown). After 1 h of incubation at the
concentration of honey fraction antioxidants (15
cells) which gave the highest degree of protection in the AAPH
experiment, oxidative damage was halved with 50
there was a 35% inhibition with 100
. This can be due to:
a) direct quenching of honey components against H
; b) the
ability of some antioxidant constituents to permeate the living
cell, drop into the intracellular space and spare thiol groups, or
c) activation of GSPx.
Whatever the mechanism, the results with a purified mixture of
honey antioxidants provide the first direct evidence that the pro-
tective effect of native honey in endothelial cells is basically due
to the phytochemical antioxidants in the matrix (Fig. 4 and Ta-
ble 1), acting against free radicals generated by different promo-
In addition, the presence of these phenolic acids and flavonoids
provides a reasonable explanation for the antioxidant power
we found in native multiflora honey, using different methods
The results of this study in living cells incubated with a reason-
ably low concentration of native honey and challenged with dif-
ferent free radical promoters provide unequivocal evidence that
honey possesses a wide spectrum of antioxidant activity, which
can give rise in vivo to preventive and curative effects in several
inflammatory pathologies and could play a role in cancer chemo-
prevention. A link has been demonstrated between inflamma-
tion and tumorigenesis in a mouse model of colitis-associated
cancer [11]. In addition in cancer cells honey might help prevent
proliferation by reducing the high oxidative state that cancer
cells need for proliferation [22].
The fact that native honey was an effective quencher of free radi-
cals when incorporated in endothelial cells at a 1% dilution ex-
cludes any intervention of the redox-active H
since its forma-
tion is negligible at this dilution. For the same reason, any protec-
tive effect due to the chemico-physical properties of the complex
matrix, or to the action of some trace mineral with antioxidant
properties, can be excluded.
One of the main findings reported in the present study is that the
honey components spare or regenerate endothelial GSH, which is
the first line of defence in the endothelial cells of the vascular
wall against the atherogenic action of peroxidized LDL and
against the strong oxidant peroxynitrite formed in those parts
of the arterial wall exposed to oscillatory shear stress (OSS),
where there is over-production of ROS due to up-regulation of
membrane NADPH oxidase [25]. Thus, honey components com-
ing into contact with a tissue rich in endothelial cells (such as the
arterial wall) can prevent the atherogenic action of oxidized LDL,
and boost the intracellular GSH pool which plays a key role in
Fig. 5 Protective effect of the antioxidant honey extract
from multiflora honey in endothelia cells against: (A) peroxyl
radicals (generator 10 mM AAPH, probe BODIPY) and (B)in-
tracellular ROS production from 50 and 100
DCPH). The concentration was expressed as
gTE/10 cells.
Results are expressed as the mean S.D. of 4 independent
Beretta G et al. Antioxidant and Radical ¼ Planta Med 2007; 73: 1182 ± 1189
Original Paper
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Original Paper
... of vitamin E, like γ-tocopherol. However, the most efficient antioxidative effect was gained by synergistic action of RA and vitamin E. RA probably achieves synergistic effect with vitamin E by decreasing vitamin E oxidation, rather than regeneration, like other antioxidants that act synergistically with vitamin E [30,31]. When TAS, TOS and PAB values and TAS/TOS ratio of Med(i)ra samples were compared before and after TBH addition, no significant difference was observed. ...
... In addition to studies that showed antioxidative effects of hydrosols and rosmarinic acid [11,12,14,15,20], study performed on endothelial cells showed significant antioxidative protection of honey in presence of various prooxidants. Honey protects GSH as well as regenerates GSH from GSSG, which is, most probably, effect of honeys lipophilic components [30]. ...
Disrupted balance between free radicals and antioxidants leads to a pathophysiological state of oxidative stress, which is the underlying cause of many diseases. Since some synthetic antioxidants show some potentially adverse effects, many studies aim to find natural antioxidants. Secondary plant metabolites, such as polyphenols, phenolic acids, which include rosmarinic acid, terpenes and terpenoids have antioxidative properties and can be found in different hydrosols and honey. The aim of this study is to evaluate the in vitro antioxidative activity of the supplement based on hydrosol mixture, rosmarinic acid and honey. A series of diluted (100%, 50%, and 25%) supplements were added to the serum pool, collected from healthy donors. A second series of samples was prepared with the same amounts of supplement and addition of tert-Butyl hydroperoxide (TBH) as a prooxidant. Alongside these samples, sera samples with Trolox (hydrophilic vitamin E analog) and Trolox with TBH were prepared. All these samples were tested for the following oxidative stress parameters: total antioxidative status (TAS), paraoxonase-1 (PON-1), total sulfhydryl groups content (SHG), total oxidative status (TOS), prooxidative-antioxidative balance (PAB) and advanced oxidation protein products (AOPP). TAS/TOS ratio was calculated as a quantitative measurement of antioxidative and oxidative substances ratio in the serum. Antioxidative parameters TAS, SHG, TAS/TOS ratio and prooxidant/antioxidant balance parameter PAB, were not significantly different for the supplemened samples with and without the addition of TBH, which indicates that the supplement keeps its antioxidative properties, despite the addition of TBH. The supplement showed significant antioxidative properties as a result of the synergistic effect of rosmarinic acid, terpenes, and terpenoids from hydrosols and polyphenols and other antioxidant substances from honey.
... Regarding scientific literature, to the best of our knowledge, no author has explored the effect of avocado honey against oxidative stress in vitro or in vivo. Several investigations have described the antioxidant activity of other types of honey (gelam, multifloral, or manuka honey) in vitro against different stressors such as AAPH [66], lipopolysaccharide [67], and bovine thrombin [68]. In accordance with the in vivo studies, some authors showed promising results of these other types of honey against oxidative stress in C. elegans [23] and rodents [69][70][71], but some of them did not directly address ROS-related measurements [69,71]. ...
... against different stressors such as AAPH [66], lipopolysaccharide [67], and bovine thrombin [68]. In accordance with the in vivo studies, some authors showed promising results of these other types of honey against oxidative stress in C. elegans [23] and rodents [69][70][71], but some of them did not directly address ROS-related measurements [69,71]. ...
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There is growing evidence that Alzheimer’s disease (AD) can be prevented by reducing risk factors involved in its pathophysiology. Food-derived bioactive molecules can help in the prevention and reduction of the progression of AD. Honey, a good source of antioxidants and bioactive molecules, has been tied to many health benefits, including those from neurological origin. Monofloral avocado honey (AH) has recently been characterized but its biomedical properties are still unknown. The aim of this study is to further its characterization, focusing on the phenolic profile. Moreover, its antioxidant capacity was assayed both in vitro and in vivo. Finally, a deep analysis on the pathophysiological features of AD such as oxidative stress, amyloid-β aggregation, and protein-tau-induced neurotoxicity were evaluated by using the experimental model C. elegans. AH exerted a high antioxidant capacity in vitro and in vivo. No toxicity was found in C. elegans at the dosages used. AH prevented ROS accumulation under AAPH-induced oxidative stress. Additionally, AH exerted a great anti-amyloidogenic capacity, which is relevant from the point of view of AD prevention. AH exacerbated the locomotive impairment in a C. elegans model of tauopathy, although the real contribution of AH remains unclear. The mechanisms under the observed effects might be attributed to an upregulation of daf-16 as well as to a strong ROS scavenging activity. These results increase the interest to study the biomedical applications of AH; however, more research is needed to deepen the mechanisms under the observed effects.
... The KC was used five lit by using a smoking machine for fifteen minutes and exposing the rats to the sidestream of the KC for five minutes, then the rats were rested to ten minutes and ventilation by removing the box cover. This operation was repeated five times a day for four weeks, where the rats were exposed to the sidestream of the KC for six days in a week [18], [19]. ...
Conference Paper
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The aim of this study was to investigate the effects of exposure cigarette smoke on the cardiac tissues in male rats and the improvement role of Sidr honey. Twenty eight male rats were divided into four groups; Group 1: control rats; group 2: rats were given Libyan Sidr honey (100 mg/kg b.w./d.) orally for 4 weeks.; group 3: rats were exposed to the five lit from sidestream of the Karelia red cigarettes (5 times/d.) by a machine smoking for 4 weeks.; and group 4: rats were received the Sidr honey (100 mg/kg b.w./d.) orally for 2 weeks, then the rats were exposed to the Karelia cigarettes generated by a machine smoking with given the Sidr honey for 4 weeks. The X-Ray radiography of rats showing, heart mildly enlarged size in the KC-exposed rats as compared with the NC rats. While, the POR rats showed normal heart size when compared with KC rats. Moreover, the KC group showed a significant increase (P < 0.05) in CK, CK-MB, and LHD as compared to the NC group, whereas the POR group showed a significant decrease (P < 0.05) in the CK, CK-MB, and LHD when compared with the KC group. Histological investigation of the heart tissues of the KC group showed different histopathological changes as compared to the NC group. Nevertheless, the POR group showed the marked improvement in the heart tissues as compared to the KC rats. Conclusion, results demonstrated that Libyan Sidr honey significantly reduced the toxic effects of KC-exposed on the heart structures.
... Many studies have demonstrated that particular physicochemical characteristics and mineral content in various locations across the world, together with chemometric analysis, can be a valuable tool in determining the botanical or geographical origin of honey that enters the market. Moisture, sugar, hydroxy-methyl furfural (HMF), mineral composition, and a variety of other factors influence honey quality in terms of both sugar and microbiological qualities (Beretta, et al. 2007,Gasparrini, et al. 2017. ...
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This article examines the physicochemical and nutritional characteristics of honey produced in different regions of Kosovo. A total of 26 samples were gathered from various locations, and beekeepers were interviewed about their honey-production techniques and bee-feeding practices. The samples underwent analysis to determine parameters such as moisture content, total solids, pH, acidity, ash content, proteins, conductivity, and brix%. Notably, the physicochemical properties of honey differed significantly across the regions. Moisture content ranged from 15.02% to 18.80%, with the lowest found in Ferizaji and the highest in Sharri. Brix% concentration at 20°C varied from 79.50% to 82.60%, with Sharri exhibiting the lowest and Prishtina displaying the highest value. The acidity and pH levels of all honey samples were measured between 4.97 and 5.63, and 3.56 to 5.60, respectively. Additionally, the Pfund scale was employed to evaluate the color of the honey, indicating white hues for Sharri, extremely light white or white for Prishtina and Ferizaj, and exceptionally white for Skenderaj. This study concludes that geographical location, feeding systems, nectar sources, honey age, and beekeeper processing methods significantly impact the physicochemical and nutritional properties of honey, including its color.
... Natural honey has been reported to prevent lipid peroxidation in the myocardium in vivo (Afroz et al. 2016a;Khalil et al. 2015). These synergistic radical scavenging effects of natural honey may be mediated by both the enzymatic and nonenzymatic antioxidants that are involved in the cardiovascular defense mechanisms (Beretta et al. 2007;Bt Hj Idrus et al. 2020;Olas 2020;Rakha et al. 2008). ...
... Erythritol does not promote tooth decay and does not cause gastric side effects like other sugar alcohols. [20] Obesity and metabolic syndrome: A 2003 technical report by the World Health Organization (WHO) provides evidence that high intake of sugary drinks (including fruit juice) increases the risk of obesity by adding to overall energy intake. By itself, sugar is not a factor causing obesity and metabolic syndrome, but ratherwhen over-consumedis a component of unhealthy dietary behavior. ...
... To address this issue, some researchers have attempted to reduce sugar interference in honey analysis. Beretta et al. [18] employed an acidic version of the FC assay, as suggested by Vinson [13,14], whereas Šarić et al., utilized PVPP as a pre-assay clean-up step prior to TPC analysis [19]. Finally, Nakaya et al. [20] utilized phenolic honey extracts in their TPC determination. ...
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The Folin-Ciocalteu assay is a widely used method for measuring the total phenolic content (TPC) in honey, but it can be affected by the presence of reducing sugars in honey, which can lead to interference and an over-estimation of its TPC. To optimize the Folin-Ciocalteu assay for honey analysis, the effect of pH on the assay was investigated. A number of pH scenarios were tested using different concentrations of Na2CO3 (0.00%, 0.75%, 0.94%, and 7.50%) in order to minimize reducing sugar interference and maximize the reaction of phenolics in the assay. The modified TPC method was then validated in accordance with current International Council on Harmonisation (ICH) guidelines. The findings of this study demonstrate that the traditional Folin-Ciocalteu assay (using 7.50% aqueous Na2CO3 solution, pH 10.8) leads to a significant overestimation of the TPC of honey due to the interference of reducing sugars. However, a pH of 7.9, achieved by using a 0.75% aqueous Na2CO3 solution, provides suitable conditions to account for most of the phenolic compounds without interference from reducing sugars. This finding was further confirmed by testing various sugar solutions and artificial honey which yielded TPC values below the established limit of detection and quantification of the assay. However, a slight increase in pH, even by a moderate deviation (pH 8.9), leads to significant discrepancies in absorbance readings, indicating that pH control is crucial for the accurate analysis of TPC in honey.
... The antimicrobial activity of honey is because honey does not support the growth of yeast and bacteria (Voidarou et al. 2011). The factors responsible for this antimicrobial activity of honey are the enzymatic glucose oxidation reaction, high osmotic pressure/low water activity, low pH/acidic environment, low protein content, high carbon-to-nitrogen ratio, low redox potential due to the high level of reducing sugars, a viscosity that limits dissolved oxygen and other chemical agents/phytochemicals, such as glucose oxidase, and hydrogen peroxide (Beretta et al. 2007) The peroxidases and many chemical products with low antibacterial level were antimicrobial agents in honey, and this includes terpenes, pinocembrin, benzyl alcohol, 3,5-dimethoxy-4-hydroxybenzoic acid (syringic acid), methyl-3,5-dimethoxy-4-hydroxybenzoate (methyl syringate), 2-hydroxy-3-phenylpropionic acid, 2-hydroxybenzoic acid, 3,4,5-trimethoxybenzoic acid and 1,4-dihydroxybenzene (Obi et al. 1994). ...
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Background Honey is consumed for the derived benefits as sweetener, for healing wounds, skin burns, regulating blood sugar level, treatments of some ailments, etc. In Nigeria, adulteration of honey is a serious concern, and these adulterated or fake honeys have health implications. This research aims to compare and contrast the physicochemical qualities, functional health and medicinal values of honey samples from local honey parkers, supermarkets and beekeepers’ sales outlets in Southwest Nigeria. Results The darkness of the honey from the three sources was significantly different. The honey samples from the beekeepers have ash contents values of 0.25–1.0 in line with CAC and IHC, whereas 7(46.66%) and 2(13.33%) of honey samples from the supermarkets and local handlers/parkers, respectively, deviated from this standard values. The wound healing development after 8 days of topical treatment of incision wounds with honey from beekeepers shows a good development. 93.3% of the honeys sourced from the beekeepers, 58.06 and 66.05% of the honeys sourced from the supermarkets, and the local honey markets were found to be original. Conclusion The originality of honey for consumers’ satisfaction is dependent on the complimentary factors of physicochemical properties, functional health and medicinal values. These findings provide information for consumers’ awareness on categorization of honey as original/pure, adulterated and fake and the health implications.
... Toxic to many microbes. During the ripening of honey, glucose oxidase is in activated but regains its activity if the honey is diluted [21]. Revamil honey produced 3.47 0.25 mm H 2 O 2 40% (v/v) honey after 24 hours, but no H 2 O 2 detectable in the Manuka honey they tested, suggesting that non peroxide factors are responsible for the antimicrobial activity of Manuka honey. ...
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Honey has been used in wound dressing for thousands of years, but only in more recent times has a scientific explanation become available for its effectiveness. The principle aim and objectives of this current work on honey was to confirm this assertion on the usefulness and medicinal value of honey. It also probed the broad-spectrum of antibacterial activity of Honey however we observed that there was much variation in potencies between different honeys from diverse sources. In this context, Gelam, Tualang and Manuka honey is used on wound complication to assess their healing potentials in which each of the honeys are applied on dressing before put on the affected lesions on the skin. Statistical analysis such as chi- square (χ2) test is used to compare association of diverse honey varieties which shows that the differences between honey varieties are not significant (P<0.05). Glucose oxidase is induced but regain its activity if the honey is diluted. It can be concluded from in vitro studies that honey has powerful antimicrobial and anti-inflammatory activities against dermatologically relevant microbes.
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Treatments for prostate cancer, cervical cancer, colorectal cancer and liver cancer often include surgical and/or radiotherapy treatments that lead to poor quality of life. The studies to find potential natural anticancer sources for use in the treatment of cancer types are actively being pursued in the world. This study aimed to investigate the cytotoxic effects of eucalyptus honey collected from Turkey on selected cancer cell lines. In this study, human prostate cancer cell line PC3, cervical cancer cell line HeLa, colorectal cancer cell line CaCo2 and liver cancer cell line HuH7 were used. Following the application of eucalyptus honey at various concentrations (0.78% - 50%) to the cell lines for 48 and 72 hours, cell viability assay (MTT) was performed to determine the number of viable cells that was indicative of cytotoxicity eucalyptus honey. The concentrations in which the cell lines lose heir viability by 80% or more by exposure to eucalyptus honey, and the viability rates of the cells at these concentrations were considered. The results showed that 6.25% concentration of the eucalyptus honey resulted in 7.6% and 7.7% viability at 48 and 72 hours for HeLa cell line, respectively. Moreover, the viability ratios of 20.7% for CaCO2 cell line at 72 hours at a concentration of 12.5% eucalyptus honey; the viability of 5.3% and 5.1% at 48 and 72 hours for HuH7 cell line, the viability of 5.2% at 72 hours for PC3 cell line, the viability of 20.4% at 48 hours of CaCO2 cell line; at 25% eucalyptus honey concentration and the viability of 7.7% in 48 hours of PC3 cell line were determined.
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The aim of this work was to establish a solid platform of analytical information for the definition/standardization of the antioxidant properties of honey. We investigated first the antioxidant/radical scavenging capacity of 14 commercial honeys of different floral and geographic origin, using a battery of spectrophotometric tests: Folin-Ciocalteu assay for phenol content (PC), ferric reducing antioxidant power (FRAP assay) for total antioxidant activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay for antiradical activity, absorbance:450 (ABS450) for color intensity and one fluorimetric method: ORAC, oxygen reactive antioxidant capacity for the antilipoperoxidant activity. Then the data from different procedures were compared and analysed by multivariate techniques (correlation matrix calculation, principal component analysis (PCA)). Significant correlations were obtained for all the antioxidant markers (r ranging from 0.933 to 0.716), with antioxidant properties strictly correlated to the phenolic content and honey color intensity. PCA found different clusters of honey based on the antioxidant power and very likely also on chemical composition. The results of this study demonstrated that only through a combination of antioxidant testings, comparative analyses, and chemometric evaluation we can achieve a strictly rigorous guideline for the characterization of the antioxidant activity of honey, an invaluable tool for the understanding/demonstration of its antioxidants linked therapeutic efficacy.
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We investigated the effects of water-soluble derivative of propolis (WSDP), caffeic acid, honey, royal jelly and bee venom on tumour development and metastasis in murine tumour models. Transplantable murine tumours were used: a spontaneous mammary carcinoma (MCa) and a methylcholanthrene–induced fibrosarcoma (FS) of CBA mouse. Metastases in the lung were generated by injecting 105 or 2 × 105 viable tumour cells intravenously. Tumours in the hind leg were generated by subcutaneous injection of 104 or 105 mammary carcinoma cells. Oral application of WSDP or caffeic acid significantly reduced subcutaneous tumour growth and prolonged survival of mice. Honey also exerted a pronounced antimetastatic effect (p < 0.01 or p < 0.001) when applied before tumour cell inoculation (2 g kg−1 orally once a day for 10 consecutive days). Royal jelly did not affect the formation of metastases when given intraperitoneally or subcutaneously. However, synchronous application of tumour cells and royal jelly intravenously significantly (p < 0.001) inhibited the formation of metastases. When bee venom was injected intratumourally, tumours decreased in size. These findings demonstrated that honey-bee products given orally or systemically may have an important role controlling tumour growth and metastasis. Copyright © 2004 Society of Chemical Industry
Hypothesis Tumor implantation (TI) development at the surgical wound following cancer surgery is still an unresolved concern. Trocar site recurrence, which is likely a form of TI, has become one of the most controversial topics and, with the widespread acceptance of laparoscopic surgery, has caused renewed interest in questions about TI. Honey has positive effects on wound healing. Physiological and chemical properties of honey might prevent TI when applied locally.Design, Interventions, and Main Outcome Measures Sixty BALB/c strain mice, divided into 2 groups, were wounded in the posterior neck area. Group 1 mice formed the control group, and group 2 mice had wounds coated with honey before and after tumor inoculation. All wounds were inoculated with transplantable Ehrlich ascites tumor. The presence of TI was confirmed in the wounded area by histopathological examination on the 10th day.Results Tumor implantation was achieved in all group 1 animals and verified by palpable mass and histopathological examination. In group 2 mice, although TI could not be detected macroscopically, it was revealed by pathological examination in 8 cases. Tumor implantation was less likely in group 2 mice (8 of 30 vs 30 of 30; P<.001).Conclusions Tumor implantation was markedly decreased by the application of honey pre- and postoperatively. It is possible that the physiological and chemical properties of honey protected wounds against TI. Honey could be used as a wound barrier against TI during pneumoperitoneum in laparoscopic oncological surgery and in other fields of oncological surgery.
This paper describes several methods for isolation of the antibacterially active phenolic fraction of honey derived from the native New Zealand manuka tree, Leptospermum scoparium (Myrtaceae). This fraction consists of phenolic derivatives of benzoic acids, cinnamic acids and flavonoids, all of which have been identified previously in honeys which do not exhibit non-peroxide residual antibacterial activity. The flavonoids had not previously been identified in manuka honey. Furthermore, the flavonoids were different from those found in the leaves of manuka trees but were the same as those found in European honeys and propolis. While most of these phenolic products possess antibiotic activity, they do not individually or collectively account for the antibacterial activity of `active' manuka honey. Essentially all of this activity is associated with the carbohydrate fraction of the honey.
Twenty flavonoid aglycones from honey were analysed by HPLC on reversed-phase columns. Different solvents were used in order to optimize the detection of those flavonoids which could be considered as markers for the floral origin of honey. None of the solvent systems used allowed the resolution of all the flavonoids from honey included in this analysis. The different solvent systems were then applied to the analysis of flavonoids from citrus and rosemary honeys. The methanol-water system permitted the separation of hesperetin, the marker of citrus honey, whereas the acetonitrile-water system was the best for the separation of all the flavanones and the detection of apigenin, the marker of rosemary honey. The presence of the flavone techtochrysin was also demonstrated in both honeys. The use of a diode-array detector proved very useful for studies of the floral origin of honey by HPLC flavonoid analysis.
A new technique for the analysis of flavonoids in honey has been developed. This uses filtration of honey through Amberlite-XAD-2 and purification of the flavonoid fraction by Sephadex LH-20. The flavonoid fraction is then analysed by HPLC. This technique allowed the identification of 16 flavonoids in honey, namely quercetin, kaempferol, 8-methoxykaempferol, quercetin 3-methyl ether, isorhamnetin, kaempferol 3-methyl ether, quercetin 3,3-dimethyl ether, quercetin 3,7-dimethyl ether, galangin, luteolin, apigenin, genkwanin, chrysin, luteolin 7-methyl ether, pinocembrin and pinobanksin. The flavonoids present in ten samples of honey from La Alcarria have been HPLC analysed by this technique. The fact that the flavonoid patterns are very similar, suggests that samples from other areas should be examined in order to assess if this procedure could be useful as an adjunct in studies of the geographical origin of honey.
Several natural products are collected or manufactured by bees to construct their hive and produce honey. These include beeswax, flower volatiles, nectar, pollen, propolis and honey itself. Some of the components of these materials possess antibacterial properties and are discussed briefly to ascertain their contribution to the antibacterial activity of honey. New Zealand's manuka honey is known to possess a high level of “non-peroxide” antibacterial activity and research to identify the origin of this activity is briefly reviewed. Finally a hypothesis is advanced to explain the phenomenon of “non-peroxide” antibacterial activity in honey. The author concludes that this activity should be interpreted as residual hydrogen peroxide activity, which is probably due to the absence of plant-derived catalase from honey, an idea first suggested by Dustman in 1971. [Dustman, J. H. (1971). Über die Katalaseaktivität in Bienenhonig aus der Tracht der Heidekrautgewächse (Ericaceae). Zeitschrift für Lebensmittel-Untersuchung und Forschung, 145, 292–295]
The protein contents in honey samples of different floral origins, commercialized in several states of Brazil, were determined using the method of Bradford. The spectra of pollen of the honeys collected in those areas were studied, in order to establish the correlation between the different botanical species and the protein contents. The physicochemical properties of the honeys (colour, moisture, pH and acidity, lund test, lugol test, diastase index, reducing and non-reducing sugars and hydroxymethylfurfural contents) were also determined. The colorimetric determination of the protein content of honey samples, using the method of Bradford, was shown to be efficient and it allowed the detection of elevated protein in honey samples of Borreria verticillata, known in Brazil as “vassourinha”, from Piauı́ State.