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Honey as a Source of Dietary Antioxidants: Structures, Bioavailability and Evidence of Protective Effects Against Human Chronic Diseases

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In the long human tradition honey has been used not only as a nutrient but also as a medicine. Its composition is rather variable and depends on the floral source and on external factors, such as seasonal, environmental conditions and processing. In this review, specific attention is focused on absorption, metabolism, and beneficial biological activities on human health of honey compounds. Honey is a supersaturated solution of sugars, mainly composed of fructose (38%) and glucose (31%), containing also minerals, proteins, free amino acids, enzymes, vitamins and polyphenols. Among polyphenols, flavonoids are the most abundant and are closely related to its biological functions. Honey positively affects risk factors for cardiovascular diseases by inhibiting inflammation, improving endothelial function, as well as the plasma lipid profile, and increasing low-density lipoprotein resistance to oxidation. Honey also displays an important antitumoral capacity, where polyphenols again are considered responsible for its complementary and overlapping mechanisms of chemopreventive activity in multistage carcinogenesis, by inhibiting mutagenesis or inducing apoptosis. Moreover, honey positively modulates the glycemic response by reducing blood glucose, serum fructosamine or glycosylated hemoglobin concentrations and exerts antibacterial properties caused by its consistent amount of hydrogen peroxide and non-peroxide factors as flavonoids, methylglyoxal and defensin-1 peptide. In conclusion, the evidence of the biological actions of honey can be ascribed to its polyphenolic contents which, in turn, are usually associated to its antioxidant and anti-inflammatory actions, as well as to its cardiovascular, antiproliferative and antimicrobial benefits.
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Honey as a Source of Dietary Antioxidants: Structures, Bioavailability and Evidence
of Protective Effects Against Human Chronic Diseases
Josè M. Alvarez-Suarez, Francesca Giampieri and Maurizio Battino*
Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. Biochimica, Universita Politecnica delle Marche, Via
Ranieri 65, 60100 Ancona, Italy
Abstract: In the long human tradition honey has been used not only as a nutrient but also as a medicine. Its composition is rather variable
and depends on the floral source and on external factors, such as seasonal, environmental conditions and processing. In this review, spe-
cific attention is focused on absorption, metabolism, and beneficial biological activities of honey compounds in human. Honey is a super-
saturated solution of sugars, mainly composed of fructose (38%) and glucose (31%), containing also minerals, proteins, free amino acids,
enzymes, vitamins and polyphenols. Among polyphenols, flavonoids are the most abundant and are closely related to its biological func-
tions. Honey positively affects risk factors for cardiovascular diseases by inhib iting inflammation, improving endothelial function, as
well as the plasma lipid profile, and increasing low-density lipoprotein resistance to oxidation. Honey also displays an important antitu-
moral capacity, where polyphenols again are considered responsible for its complementary and overlapping mechanisms of chemopre-
ventive activity in multistage carcinogenesis, by inhibiting mutagenesis or inducing apoptosis. Moreover, honey positively modulates the
glycemic response by reducing blood glucose, serum fructosamine or glycosylated hemoglobin concentrations and exerts antibacterial
properties caused by its consistent amount of hydrogen peroxide and non-peroxide factors as flavonoids, methylglyoxal and defensin-1
peptide. In conclusion, the evidence of the biological actions of honey can be ascribed to its polyphenolic contents which, in turn, are
usually associated to its antioxid ant and anti-inflammatory actions, as well as to its cardiovascular, antiproliferative and antimicrobial
benefits.
Keywords: Antioxidant capacity, antimicrobial action, bioavailability, cancer, cardiovascular disease, honey.
INTRODUCTION
It is known that oxidative stress, caused by an imbalance be-
tween the production of highly reactive molecules and antioxidant
defences, causes structural and functional damage to lipids, proteins
and nucleic acids leading to many biological complications includ-
ing carcinogenesis, aging and atherosclerosis [1, 2]. There-
fore, exogenous antioxidants from diet can counteract the deleteri-
ous effects of free radicals, reducing oxidative damage [3]. Many
epidemiological studies show in fact that a diet rich in polyphenols
is often associated with a lower incidence of several chronic pa-
thologies, such as obesity, infections, cardiovascular and neurologic
diseases, and cancer [4-6]. In the long human tradition, honey has
been used not only as a sugar but also as a medicine: it has been
employed in many cultures for its medicinal properties, including as
a remedy for burns, cataracts, ulcers and wound healing [7, 8]. Only
recently, scientific research focused its attention on the therapeutic
effects of honey, in particular on its capacity to protect against car-
diovascular diseases [9, 10], cancer [11-14] and microbial infec-
tions [15, 16]. These health-p rotective and therap eutic impacts of
honey depend on the presence of various antioxidant components,
especially phenolic compounds, such as flavonoids and phenolic
acids, most of which express relevant antimicrobial, antioxidant,
anti-inflammatory, antimutagenic activities capacities both in vi-
tro and in vivo [17]. Besides phenolic compounds and sugars,
honey is a source of proteins, free amino acids, minerals, enzymes,
and vitamins, representing therefore a good healthy choice.
This review focuses on the nutrient and phytochemical contents
of honey and on its antioxidant capacity. An overview on the
bioavailability and metabolism of the most abundant honey phyto-
chemicals after consumption is also presented, and the currently
hypothesized health benefits related to honey consumption is re-
viewed, with particular attention given to recent evidence on its
impact on cardiovascular health, cancer prevention, hyperglycemias
regulation and antimicrobial activity.
*Address correspondence to this autho r at the Dipartimento di Scienze Clinich e Spe-
cialistiche ed Odontostomatologiche, Sez. Biochimica, Facoltà di Medicina. Universtà
Politecnica delle Marche, Italy, Via Ranieri 65, 60100 Ancona, Italy; Tel: +39 071
2204646; Fax: +39 071 2204123; E-mail: m.a.battino@univpm.it
1. COMPOSITION
The composition of honey is rather variable, depending on the
floral source and other external factors, such as seasonal and envi-
ronmental conditions and processing. Honey contains a variety of
approximately 180 compounds, such as sugars, proteins, free amino
acids, essential minerals, vitamins and enzymes as well as a wide
range of polyphenolic phytochemicals. This range of compounds
will be discussed below, with specific focus on the most significant
components with beneficial effect on human health, essentially
flavonoids and phenolic acids.
1.1. Nutrients
Concerning its nutrient profile (Table 1), honey represents an
interesting source of natural macro and micronutrients. First of all,
it is an important source of calories, since 100 g of honey provide
approximately 300 kcal and a daily dose of 20 g covers about 3% of
the recommended daily intake of energy (RDI)[7]. Carbohydrates
represent 95% of its dry weight: approximately a total of 26 sugars
(mono- and disaccharides) have been identified in honey (Table 2)
[18] with fructose (~ 40%) and glucose (~ 30%) as major sugars. It
is important to note that most of these sugars do not occur in nectar,
because they are the results of enzymes added by the honeybee
during the ripening of honey or by chemical action in the concen-
trated form. To a lesser extent, honey contains proteins (roughly
0.5%), mainly enzymes and free amino acids. The amount of nitro-
gen in honey is low, approximately 0.04%, though it may reach
0.1% with 40 to 65% as protein form and the rest as free amino
acids. The total of free amino acids in honey corresponds approxi-
mately to 1% (w/w) of total nitrogen and ranges between 10 and
200 mg/100 g, according to its origin (nectar, honeydew or floral
origin), with proline as the major contributor, corresponding to
approximately 50% of total free amino acids [19]. Since pollen is
the main source of honey amino acids, their profile could be charac-
teristic and indicative of their botanical origin [20]. In addition to
classical amino acids also b-alanin e (b-Ala), a-alanine (a-Ala), g-
aminobutyric acid (Gaba) and ornithine (Orn) have been found and
identified in honey [19-22].
Honey also presents a variable number of mineral elements,
which varies according to geographic region, soil type and floral
2 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
Tabl e 1. Chemical Composit ion of Honey*.
Proximates and carbohydrates Mineral content Vitamins content
Water (g) 17.1 Calcium (mg) 4.4-9.20 Ascorbic Acid (C) (mg) 2.2 -2.4
Energy (kcal) 304 Potassium (mg) 13.2-16.8 Thiamin (mg) < 0.006
Carbohydrates (total) (g) 82.4 Copper (mg) 0.003-0.10 Riboflavin (mg) < 0.06
....Fructose ( g) 38.5 Iron (mg) 0.06-1.5 Niacin (m g) < 0.36
....Glucose ( g) 31.0 Magnesiu m (mg) 1.2-3.50 Pantothe nic acid (mg) < 0.11
....Maltose (g ) 7.2 0 Ma nganese (mg) 0.02-0.4 Pyridoxine (B6) (m g) < 0.32 m g
....Sucrose ( g) 1.50 Phosphoru s (mg) 1.9 -6.30
Proteins, amino acids,
vitamins and minerals (g) 0.50 Sodium (mg) 0.0-7.60
Zinc (mg) 0.03-0.4
Se (g) 1.0-2.91
*Amount in 100 g of honey
Table 2. Carbohydrates Composition in Honey[18].
Trivial nomenclature Systematic nomenc lature
Disaccharide
Cellobiose 2 O--D-glucopyranosyl-(1 -> 4)-D-glucopyranose
Gentiobiose 2 O--D-glucopyranosyl-(1 -> 6)-D-glucopyranose
Isomaltose 2 O--D-glucopyranosyl-(1 -> 6)-D-glucopyranose
Isomaltulose 4 O--D-glucopyranosyl-(1 -> 6)-D-frutofuranose
Kojibiose 1 O--D-glucopyranosyl-(1 -> 2)-D-glucopyranose
Laminaribiose 3 O--D-glucopyranosyl-(1 -> 3)-D-glucopyranose
Leucrose 4 O--D-glucopyranosyl-(1 -> 5)-D-fructofuranose
Maltose 1 O--D-glucopyranosyl-(1 -> 4)-D-glucopyranose
Maltulose 2 O--D-glucopyranosyl-(1 -> 4)-D-fructose
Melibiose 4 O--D-galactopyranosyl-(1 -> 6)-D-glucopyranose
Neo-trehalose 3 O--D-glucopyranosyl--D-glucopyranoside
Nigerose 2 O--D-glucopyranosyl-(1 -> 3)-D-glucopyranose
Palatinose 2 O--D-glucopyranosyl-(1 -> 6)-D-fructose
Saccharose 1 O--D-glucopyranosyl--D-frutocfuranoside
Turanose 1 O--D-glucopyranosyl-(1 -> 3)-D-fructose
Trisaccharide
kestose 4 O--D-glucopyranosyl-(1 -> 4)-O--D-glucopyranosyl-(1 -> 2)-D-glucopyranose
1-kestose 4 O--D-glucopyranosyl-(1 -> 2)--D-fructofuranosyl-(1 -> 2)--D-fructofuranoside
Erlose 1 O--D-glucopyranosyl-(1 -> 4)--D-glucopyranosyl--D-fructofuranoside
Isomaltotriose 2 O--D-glucopyranosyl-(1 -> 6)-O--D-glucopyranosyl-(1 -> 6)-D-glucopyranose
Isopanose 2 O--D-glucopyranosyl-(1 -> 4)-O--D-glucopyranosyl-(1 -> 6)-D-glucopyranose
Laminaritriose 4 O--D-glucopyranosyl-(1 -> 3)-O--D-glucopyranosyl-(1 -> 3)-D-glucopyranose
Maltotriose 2 O--D-glucopyranosyl-(1 -> 4)-O--D-glucopyranosyl-(1 -> 4)-D-glucopyranose
Melezitose 2 O--D-glucopyranosyl-(1 -> 3)-O--D-fructofuranosyl-(2 -> 1)--D-glucopyranoside
Panose 2 O--D-glucopyranosyl-(1 -> 6)-O--D-glucopyranosyl-(1 -> 4)-D-glucopyranose
Rafinose 2 O--D-galactopyranosyl-(1 -> 6)-O--D-glucopyranosyl--D-fructofuranoside
Teanderose 2 O--D-glucopyranosyl-(1 -> 6)--D-glucopyranosyl--D-fructofuranoside
1- Majority 2- Mino rity 3- Traces 4- not con firmed
origin, representing approximately 0.2% of its dry weight. The
amount of minerals and trace elements in honey is small and their
contribution to the RDI is margin al (Table 1). Minerals and trace
elements play a key role in biomed ical activities associated with
food, since these elements have a multitude of known and unknown
biological functions, such as the maintenance of intracellular oxida-
tive balance [23, 24]. Recently, the presence of Se content in Portu-
guese unifloral honeys was reported [25]. Se is an essential trace
element, and its role in the metabolism is largely related to its in-
corporation into selenoproteins [26]. As an essential component of
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 3
antioxidant enzymes GSH-Px and thioredoxin reductase, this ele-
ment may promote endogenous enzymatic capacity to protect
against excessive generation of free radicals [27]. Honey also con-
tains choline (0.3-25 mg/kg) and acetylcholine (0.06-5 mg/kg).
Choline is essential for cardiovascular and brain function as well as
for cellular membrane composition and repair, while acetylcholine
acts as a neurotransmitter [7].
Finally, the vitamin content in honey is low. Vitamins such as
thiamin (B1), riboflavin (B2), pyridoxin (B6) and niacin have been
reported in honey but in general their amount is small and the cor-
responding contribution of honey to the RDI is very limited [7, 8].
1.2. Enzyme and Organic Acids
One of the characteristics that distinguish honey from all other
sweetening agents is the p resence of enzymes. Th ey may origin ate
from the bee, pollen, nectar, even from yeasts or micro-organisms
present in honey. Three main enzymes can be found: invertase,
diastase and glucose oxidase. Invertase splits sucrose releasing its
simple constituents; during this action, other groups of more com-
plex sugars have been found in small amounts, explaining in part
the complexity and variability of the minor sugars of honey. Inver-
tase remains in honey and retains its activity for some time, com-
pleting its activity when honey is ripened. Despite this, the sucrose
content of honey never becomes zero. Since invertase also synthe-
sizes sucrose, the final low value for the sucrose content of honey
probably represents an equilibrium between splitting and forming
sucrose, an aspect which is often taken into account when measur-
ing the maturity and quality of honey [28]. Diastase or amylase
digests starch to simpler compounds. The origin of this enzyme in
honey is controversial and it is not known for sure if it comes from
nectar, pollen or bee, or what its functions are because starch is not
present in honey. Alpha-amylase randomly breaks the down starch
chains, producing dextrins and beta-amylase which divides the
reducing sugar maltose from the terminal starch chains. Diastase
activity is used as an important indicator of honey quality: the
higher the content of this enzyme, the higher is the quality of honey
[28]. Despite its discrete contribution to the human diet, the sup-
plementation of diastase by honey can be interesting and helpful in
increasing the metabolism of sugars, especially related with carbo-
hydrate digestive disorders. Finally, glucose oxidase (GOx) is of
interest because it is related to honey antibacterial properties. GOx
converts dextrose to a related compound, a gluconolactone, which
in turn forms gluconic acid, the principal acid in honey, and hydro-
gen peroxide the main agent responsible for antibacterial activity in
most honeys. GOx amount varies in different honeys and since it
was found in the pharyngeal gland of the honey bee this is probably
the most likely source of this enzyme [28, 29]. Other enzymes re-
ported in honey are catalase and acid phosphatase. It is important to
note that honey enzymes can be destroyed or weakened by heat
caused by careless handling during industrial processing or storage.
Finally, honey contains also a series of organic acids corre-
sponding to 0.17-1.17% of the total acids, representing less than
0.5% of solids [28] which contribute to the flavor and in part are
responsible for its excellent stability ag ainst microorganisms and
are also associated with honey antibacterial activity [30]. Among
these acids, glu conic acid has been id entified as the most important
one. Other organic acids in honey are formic, acetic, butyric, lactic,
oxalic, succinic, tartaric, maleic, pyruvic, pyroglutamic, a-
ketoglutaric, glycollic, citric, malic, 2- or 3-phosphoglyceric acid,
- or -glycerophosphate, and glucose 6-phosphate.
1.3. Phenolic Phytochemicals in Honey
Honey phytochemicals are mainly represented by the extensive
class of phenolic compounds depending on honey origin [31-36]
and, therefore, expected to have different biological activities and
huge biological potentialities in humans [17]. The major class of
phenolic compounds in honey is represented by flavonoids (fla-
vonols, flavanols and flavones) followed by phenolic acids (benzoic
acids, phenylacetic and hydroxycinnamic acids). The most common
phenolics acids and flavonoids identified in honey are shown in
(Table 3).
Table 3. Most Common Phenolic Acid and Flavonoids Identified in
Honey.
Phenolic acid Flavonoids
4- dimethylaminobenzoic acid Apigenin
Caffeic acid Genistein
p-coumaric acid Pinocembrin
Gallic acid Tricetin
Vallinic acid Chrysin
Syringic acid Luteolin
Chlorogenic acid Quercetin
Quercetin 3-methyl ether
Quercetin-diglycoside
Quercetin–3-O-rutinoside
Quercetin–O–rhamnoside
Kaempferol
Kaempferol 8-OMe
Kaempferol 3-OMe
Kaempferol-7-O-rhamnoside
Kaempferol-3-O-glycosyl
Kaempferol-7-O-glycosyl
Galangin
Pinobanksin
Myricetin
Myricetin 3-OMe
Myricetin 3,7,4’,5’-OMe
Phenolic phytochemicals are the largest group of phytochemi-
cals ubiquitous in plants and are incorporated into the honey via
nectar / pollen from plants visited by the honeybee. Simple phenols
are those with a C6 carbon structure (such as phenol itself, cresol
and thymol) (Fig. 1A). Phenolic acids are derived from benzoic
acid, phenylacetic and hydroxycinnamic acid (Fig. 1B, 1C and 1D,
respectively) where the hydroxyl (OH) groups can be substituted in
the aromatic ring. Some of them have a C6-C1 structure (e.g. gallic,
vanillic and syringic acids) and aldehydes (e.g. vanillin). Others
have a C6-C2 structure, such as phenylacetic acids and acetophe-
nones. Phenylpropanoid derivatives of a C6-C3 structure are mainly
represented by hydroxycinnamic acids such as p-coumaric, ferulic
and caffeic acids and their respective derivatives [37, 38]. Simple
phenols, phenolic and phenylacetic acids can be found free and
have been identified in several floral honeys,the most frequent ones
beingp-coumaric, ferulic and caffeic acids [31-36, 39]. In honey of
Leptospermum scoparium and Leptospermum polygalifolium from
New Zealand and Eucalyptus ssp. from Australia, gallic acid was
identified as the most predominant phenolic acid [40, 41]. Other
phenolic acids havealso been reported: chlorogenic, syringic, vanil-
lic and p-hydroxybenzoic acids as the agents responsible for the
antioxidant activity exhibited by the extracts of honey from differ-
ent botanical origins (Leptospermum polygalifo lium, Epilobium
angustifolium; Nyssa aquatica; Schinus terebinthifolius; Glycine
max; Melilotus spp y Robinia pseudoacacia) [39, 40].
Flavonoids are low molecular weight compounds that share a
common skeleton of diphenyl propanes, formed by two benzene
4 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
Fig. (1). Chemical structures of simple phenols with structure C6 (A), phenylacetic acid, C6-C2 (B), benzoic acids, C6-C1 (C), and hydroxycinnamic acids, C6-C3
(D).The different structures of benzoic acids and hydroxycinnamic acids are shown in C and D, respectively.
rings joined by a linear three-carbon chain (C6-C3-C6). Often, the
carbons of the propane connecting the phenyl rings may form a
closed pyran ring together with one of the benzene rings thus form-
ing a structure of 15 carbon atoms arranged in three rings, labeled
A, B and C (Fig. 2A). These compounds generally have at least
three phenolic OH groups and are generally combined with sugars
to form glycosides, with glucose as the major sugar, but also galac-
tose, rhamnose and xylose can be found, as free aglycon. At the
same time, flavonoids are further divided and classified according
to the degree of oxidation of the C ring into flavones, flavonols,
flavanones, flavanonols, flavanols or catechins, isoflavones, antho-
cyanins and anthocyanidins. Within these groups, flavonols (Fig.
2B) (e.g. quercetin, myricetin and kaempferol), flavones (Fig. 2C)
(apigenin, luteolin, diosmetin, chrysin) and flavanols or catechins
(Fig. 2D) (catechin, epicatech in, epigallocatechin, epigallo catechin
gallate) are the most abundant in honey. Flavonols are characterized
by an unsaturation between the C2 and C3 carbons of the C ring, a
ketone group in C4 and by the presence of a hydroxyl group in posi-
tion 3 of the ring, while flavones do not exhibit this latter group.
Flavanols present a hydroxyl group on carbon 3 [38, 42]. These
flavonoids may appear in the O–glycosylated form, in which one or
more hydroxyls are linked to a sugar, thus forming a O–C bond,
with glucose as the most common glycosidic unit, even if other
examples include glucorhamnose, galactose, arabinose, rutinoside
and rhamnose. Glycosylation can also occur froma direct link be-
tween the sugar and the flavonoid nucleus, forming a strong bond
C–C with the formation of C–glycosides. This type of glycosylation
occurs only at positions C6 and/or C8. The objective of glycosyla-
tion seems to form a flavonoid less reactive and more soluble in
water; the glycosylation can be seen as an essential form of protec-
tion of plants to avoid the cytoplasmic damage since flavonoids can
accumulate in vacuoles [43]. Most flavonols are in the shape of O
glycosides and rarely of C–glycosides, while the flavones are often
found in nature, both as O–glycosylated and as C– glycosylated
[44].
2. BIOAVAILABILITY AND METABOLISM OF HONEY
POLYPHENOLS
Because evidence of the potential health-promoting and dis-
ease-preventing effects of honey continues to accumulate, it is be-
coming more necessary to understand the nature of absorption and
metabolism of polyphenolic compounds, as these plays an impor-
tant role in healthy beneficial effects. Current knowledge on the
absorption and metabolism of polyphenols has been elucidated
through several in vitro methods, in situ animal experiments, and
some in vivo studies [17, 45].
Currently there are very few studies on bioavailability of honey
polyphenols in humans. The most significant study, by Schramm et
al., [46], reported that after consumption of 1.5 g of honey/kg body
of two honey types in 40 subjects, the plasma total-phenolic content
increased (P < 0.05) similarly to antioxidant and reducing capaci-
ties of plasma (P < 0 .05). These data supported the concept that
phenolic antioxidants from honey are bioavailable, and that they
increase plasma antioxidant activity by improving the defenses
against oxidative stress. However, although the honey used in this
investigation provided mg quantities of 4-hydroxybenzoic and 4-
hydroxycinnamic acids per kg of body weight, the plasma concen-
tration of these acids could not be verified by HPLC analysis. Ac-
cording to the authors this could be due to (i) less than one-third of
these compounds were absorbed, (ii) these compounds could have
been distributed quickly into body compartments other than plasma,
or (iii) the monophenols underwent first pass metabolism in the
human body.
However, the absorption of flavonoids seems much more com-
plex, fundamentally due to its chemical characteristics. Fig. (3)
illustrated the proposed mechanisms for the absorption and metabo-
lism of polyphenolic compounds in the small intestine. The avail-
able literature suggests that not only the bacterial enzymes in the
intestine [17, 47, 48], are responsible for beta-hydrolysis of sugar
moieties in the O-glycosides flavonoids. Two
-endoglucosidases
capable of flavonoid glycoside hydrolysis have also been character-
ized in human small intestine, namely lactase phlorizin hydrolase
(LPH), acting in the brush border of the small intestine epithelial
cells [48, 49] and a cytosolic
-glucosidase (CBG) as an alternative
hydrolytic step within the epithelial cells [50, 51, 52]. LPH exhibits
broad substrate specificity for flavonoid-O-
-D-glucosides, and the
released aglycone may then enter the epithelial cells as a result of
its increased lipophilicity and its proximity to the cellular mem-
brane [53]. It is has also been proposed that for CBG-catalyzed
hydrolysis to occur, the polar glycosides must be transported into
the epithelial cells, possibly with the involvement of the active so-
dium-dependent glucose transporter 1 (SGLT1) [54]. Published
studies on the bioavailability and pharmacokinetics have demon-
strated that some flavonoids can inhibit the non-Na+ -dependent
facilitated diffusion of monosaccharides in intestinal epithelial cells
[55]. Thereby, the parallel concentrative Na+ -dependent transport
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 5
Fig. (2). Chemical structures of the more common flavonoids in honey.
ATPase for monosaccharides is benefited [56]. Therefore, the two
possible routes by which the glycoside conjugates are hydrolyzed
and the resultant aglycones cross into enterocytes are LPH/diffusion
and transport/CBG [57]. To these, in the case of honey, the pres-
ence of the enzyme glycosidase in the bee salivary glands [58, 59]
should also be added producing a hydrolysis of the glycosylated
flavonoids and releasing the aglycon form. This explains, in part,
the fact that unlike other phenolics present in foods or beverages,
flavonoids in honey have been identified mostly as a form of agly-
cons and not in their glycosylated form. Phenolic aglycons are more
readily absorbed through the gut barrier than their corresponding
glycosides by passive diffusion [60] and, therefore, flavonoids pre-
sent in honey may be more readily bioavailable. It has been also
proposed that after release of the glycosides from the aglycone
about 15% of the flavonoid aglycons are absorbed with bile mi-
celles into the epithelial cells and passed on to the lymph [47, 48].
Despite this, the influence of the presence, location and structure of
the sugar moiety in the bioavailability and metabolism of glycosy-
lated flavonoids has also been highlighted [57].
Once absorbed by the intestinal epithelium and before crossing
into the bloodstream, flavonoids undergo some degree of phase II
metabolism with the generation of different conjugated products,
predominantly sulphates, glucuronides and methylated derivatives
through the action of sulfotransferases (SULTs), uridine-5`-
diphosphate glucuronosyltransferases (UGT), and catechol-
O'Methyltransferase (COMTs), respectively [57]. Besides the
metabolic biotransformation of flavonoids, which occurs by the
intestinal microflora and the gut-liver pathways, their bioavailabil-
ity and cell/tissue accumulation have been closely associated with
the multidrug-resistance-associated proteins like MRP-1 and MRP-
2 (i.e. ATP-dependent efflux transporters), also named phase III
metabolism [38] and with their tissue distribution and substrate
affinity in the various organs. It has been proposed that MRP-2,
localized on the apical membrane of cells of the small-bowel epi-
thelium, transports the already intracellular flavonol back to the
intestinal lumen, thus modulating the actual intestinal importation
of these compounds. On the contrary, MRP-1, situated on the vas-
cular pole of enterocytes, favors transport of the flavonoid from
inside the cells into the blood [61, 62]. Moreover, it is has been
proposed that MRP-3 and the glucose transporter GLUT2 are also
implicated in th e efflux of metabolites from the basolateral mem-
brane of the enterocytes [63]. Once in the portal bloodstream, me-
tabolites rapidly reach the liver: in hepatocytes, aglycones are trans-
ferred to the Golgi apparatus and possibly also to the peroxisomes,
being oxidatively degraded and subjected to further phase II me-
tabolism [57, 64]. These conjugate forms can retain their antioxi-
dant properties, while others such as quercetin quickly enter the cell
regaining their active, nonconjugated form [65, 66]. Finally, some
flavonoid conjugates with sugar moieties resistant to the action of
6 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
Fig. (3). Mechanisms for the abso rption and metabolism of flavonoid compounds in the small intestine. CBG, cytosolic
-glucosidase; COMT, catechol-O-
methyl tran sferase; GLUT2, glucose transporter; LPH, lactase phloridzin hydrolase; MRP1-2–3, multidrug-resistant proteins; Flav-Agly, Flavonoid aglycone;
Flav-Gly, Flavonoid glycoside, Flav-Met, Flavonoid sulfate/glucuronide/methyl metabolites; SGLT1, sodium-dependent glucose transporter; SULT, sulfotrans-
ferase; UGT, uridine-5´-diphosphate glucuronosyltransferase.
LPH/CBG are not absorbed in the small intestine and pass to the
colon and are therefore excreted with the faeces, while enterohe-
patic recirculation may result in som e recycling back to the small
intestine through bile excretion [67]. Others, after metabolic trans-
formation, are secreted by organic acid transporters into the blood
and subsequently excreted through the kidneys [68-70].
More studies on the bioavailability and pharmacokinetics of
polyphenols in humans are necessary. However, recently reports are
encouraging, revealing that flavonoids can be incorporated in lipo-
protein domains and plasma membranes, which generally serve as
targets for lipid peroxidation, suggesting a protective interaction of
flavonoids with lipid bilayers [50, 71] and they can also accumulate
in the nucleus [72] and mitochondria [66] affecting diverse cell
metabolic functions.
3. ANTIOXIDANT PROPERTIES
The antioxidant properties of honey have been associated to the
ability and potential of reducing oxidative reactions within the food
systems resulting as attractive/beneficial fo r human health. These
oxidative reactions can cause deleterious reactions in food products
and adverse health effects, includingchronic diseases and cancers
[73, 74]. The antioxidant capacity (AOC) has been proposed as an
indicator of the presence of beneficial bioactive compounds in
honey when it was identified as a dietary source of natural antioxi-
dants. The AOC varies greatly depending on the honey floral
source, possibly due to the differences in the content of plant sec-
ondary metabolites as polyphenolics and enzyme activities [75-80].
It has been found that several constituents of honey play a signifi-
cant role in AOC as glucose oxidase, catalase, ascorbic acid, or-
ganic acids, Maillard reaction products, amino acids, proteins, phe-
nolic acids and flavonoids. Several research groups have studied the
AOC of honey using different methods to determine alternatively
(i) the capacity to scavenge active oxygen species (e.g. the superox-
ide anion, peroxyl and hydroxyl radicals) [81, 82] and (ii) enzy-
matic or non-enzymatic capacity of lipid peroxidation inhibition
[78, 83]. Honey is a complex biological matrix that gives high vari-
ability in measurements and makes it very difficult to obtain stan-
dardized AOC indexes. Moreover, when reliable techniques are
applied, it can be demonstrated that honey has an in vitro AOC
similar to those of many fruits and vegetables on a fresh weight
basis [73]. Honey has been shown to protect food against microbial
growth [84] and deteriorative oxidation reactions, such as lipid
oxidation in meat [85], enzymatic browning of fruits and vegetables
[86], providing also an effective protection against chemically in-
duced lipid peroxidation in rat liver, brain and kidney homogenates
[78, 83]. This particular model of lipid peroxidation just cited is
interesting because it has the advantage of including and mimicking
several of the mechanisms responsible for the generation and/or
modulation of lipid peroxidation occurring in vivo: therefore, it
offers the possibility of identifying antioxidant compounds able to
mitigate lipid oxidative damage. It has been reported that honey
presents important radical scavenging cap acities (Fig. 4). Several
studies demonstrated that honey is capable of scavenging hydroxyl
and superoxide radicals [30, 78, 87-89]. The ability of honey to
scavenge free radicals and to protect against lipid peroxidation may
contribute to preventing/reducing some inflammatory diseases in
which oxidative stress is involved, offering an interesting preven-
tive and therapeutic option.
The AOC of honey, which depends on polyphenol contents, is
also correlated with its color [26, 77, 80]. Frankel et al. [90] sug-
gested that the color intensity in honey is related to pigments
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 7
(flavonoids, carotenoids, etc.). Actually, the dark honeys have
shown the highest AOC, as well as, phenolic, flavonoid and caro-
tenoid concentrations while the light-colored honeys are character-
ized to h ave the lowest valu es, with linear positive co rrelations
between color vs phenolic and flavonoid content vs radicals scav-
enging activity and protection against lipid peroxidation (P< 0.05)
[73, 75-78, 79, 80, 83, 88, 90].
Fig. (4). Radical scavenging mechanism of phenolic compounds.
3.1 Polyphenols as the Principal Contributors of Honey AOC
Since polyphenols are considered as mostly responsible for
AOC in honey, the mechanisms by which these compounds con-
tribute to its antioxidant properties are considered as the most
medically useful properties of honey. These positive characteristics
seem to be ascribed to their efficacy as metal chelators and as ex-
cellent free-radical scavengers, as well as gene modulators able to
influence enzymatic and non-enzymatic systems that regulate cellu-
lar redox balance [17, 38, 91].
Among the phenolic acids, benzoic acid is a weak antioxidant.
This capacity is increased in the case of dihydric or trihydric deriva-
tives, where the antioxidant effect depends on the relative positions
of OH groups in the aromatic ring. Thus, gallic acid (3, 4, 5-
trihydroxybenzoic acid) is the most potent antioxidant within all the
hydroxybenzoic acids. Contrary to their homologs derived from
benzoic or phenylacetic acid, hydroxycinnamic acids exhibits
greater free radical scavenging ability. This property appears to be
related to the inclusion of the unsaturated chain bonded to the car-
boxyl group as a distinctive structure which provides stability by
resonance to phenoxyl radical, even offering additional sites for the
attack of free radicals [92]. Furthermore, the existence of several
electron donor groups in the benzene ring structure (as hydroxyl or
methoxy groups in structures) also provides a greater number of
resonant structures and increases the stability of the arylic radical in
cinnamic acids, thereby favoring their antioxidant behavior.
It has been widely demonstrated that flavonoids are very effec-
tive as scavengers of reactive oxygen species (ROS) peroxyl, alkyl
peroxide, hydroxyl and superoxide radicals, as well as against reac-
tive nitrogen species (RNS) likenitric oxide and peroxynitrite, pro-
tecting against the oxidative damage induced by these molecules
[49, 93]. This activ ity is attributed basically to three chemical fea-
tures in flavonoid structure, namely an ortho-dihydroxy structure in
the B-ring [96-99], and the presence, in the C-ring, of a 2, 3 double
bond and/or of a 4-oxo function [91].
Hydroxyl groups on the B-ring donate a hydrogen and an elec-
tron to hydroxyl, peroxyl, and peroxynitrite radicals,stabilizing
them and giving rise to relatively stable flavonoid radicals. Oxida-
tion of a flavonoid occurs on the B-ring when catechol is present
[100], yielding a fairly stable ortho-semiquinone radical [101]
through facilitating electron delocalization [50]. Other hydroxyl
configurations are less clea r, as the A-ring substitu tion, where th e
increasing total number of OH groups correlates little with AOC
[91]. Moreover, the heterocyclic character of some flavonoids plays
an important role in antioxidant activity by the presence of a free 3-
OH and allowing conjugation between the aromatic rings, the
closed C-ring itself may not be critical to the activity of flavon-
oids[102]. Flavonoids with a 3-OH and 3`, 4`- catechol are reported
to be 10- fold more potent than ebselen, a known RNS scavenger
against peroxynitrite radical [91, 98]. Examples are flavonols: the
superiority of quercetin in inhibiting both metal and nonmetal-
induced oxidative damage is partially ascribed to its free 3-OH
substituent [50, 91], which is thought to increase the stability of the
flavonoid radical, while the substitution of 3-OH by a methyl or
glycosyl group decreases the AOC of this flavonol [97].
A distinguishing feature among the general flavonoid structural
classes is the presence or absence of an unsaturated 2–3 bond in
conjugation with a 4-oxo function. It has been demonstrated that
flavonoids which do not exhibit one or both features exhibit a lesser
AOC than those with both elements. The conjugation between the
A - and B rings permits a resonance effect of the aromatic nucleus
leading stability to the flavonoid radical [103]; this conjugation is
critical in optimizing the phenoxyl radical-stabilizing effect of the
3`,4`-catechol [104]. The fact that flavonols have a higher free radi-
cal scavengers capacity than flavones [104, 105] may be associated
to the greater number of hydroxyl groups and substituents 3-OH
present in their structure.
4. HONEY AND HEALTH
The biological activities of honey have long been studied using
several in vitro and animal models studies, as well as human epi-
demiologic and interventional studies ( Table 4).
4.1 Honey and CVD Risk
Studies in vitro and in vivo have shown that honey can posi-
tively affect risk factors for CVD by inhibiting inflammation, im-
proving endothelial function [106], improving the plasma lipid
profile and increasing low-density lipoprotein (LDL) resistance to
oxidation [73].
The oxidative modifications of lipoproteins play an important
role in the pathogenesis of atherosclerosis [107, 108]. In an in vitro
model a significant correlation between AOC and inhibition of
lipoprotein oxidisability from human serum by honey was reported
[73, 109], providing initial useful evidence about the protective
effect of honey against the oxidative damage of these molecules.
Besides this, the improvement of endothelial function by honey has
also been demon strated. Beretta et a l.[106] reported, using native
honey, a significative quenching activity against lipophilic cumoxyl
and cumoperoxyl radicals, with significant suppression/prevention
of cell damage, complete inhibition of cell membrane oxidation,
decrease of intracellular ROS production as well as intracellular
GSH recovery in endothelial cell cultures (EA.hy926). Moreover,
the phenolic fraction isolated from these honeys was used to pre-
treat the same endothelial cells, exposed later to peroxyl radicals
from AAPH and to hydrogen peroxide, and indicated as the main
cause of the same protective effect. In another in vitro study, re-
ported by Ahmad et a l. [110], the effect of honey on bovine throm-
bin-induced oxidative burst in human blood phagocytes was stud-
ied. The results reported that phagocytes activated by bovine
thrombin and treatment with honey showed effective suppression of
oxidative respiratory burst [110]. These results suggest that,
through the synergistic action of its antioxidants, the antioxidant
compounds present in honey could be beneficial in the interruption
of the pathological progress of cardiovascular disease,could play a
cardioprotective role and contribute to reducing the risks and effects
of acute and chronic free radical induced pathologies in vivo.
Although blood is not strictly part of the cardiovascular system,
it is in any case closely related to it functionally, and its alterations
may be a predisposing factor for CVD. Recently, an interesting area
of research which discusses the protective effect of food polyphe-
nols in red blood cells (RBC) against oxidative damage has
8 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
Table 4. Effects of Honey Consumption on Health.
Diseases Effect on health References
Cardiovascular diseases (CVD)
Reduction of cardiovascular risk factors
Inhibition of inflammation
Improvement of endothelial function
Improvement of plasma lipid profile
Increase of low-density lipoprotein (LDL) resistance to oxidation
Inhibition of Red Blood Cells (RBCs) hemolysis
Improvement of eritrocytes uptake capacity
Protection of RBCs against intracellular depletion of GSH and SOD activity
Decrease of th e susceptibility of RBCs lipid m embran e against oxidativ e damage
Maintenance of the body weight in overweight or obese subjects (no increase)
9
106
106
73
73,109
118, 120
119
118
71, 111, 113,118,120
9
Hypertension Reduction of systolic blood pressure and MDA levels
Ameliorament of susceptibility of kidneys to oxidative stress
128
129
Cancer
Antimutagenic capacity
Induc tion of apop tosis
Antiproliferative effect
Citotoxic effect on several cancer cell lines
Antimetastatic effect
145
147, 149
12, 149, 150, 152
148, 152
151
Diabetes
Reduction of glycaemia
Reduction of serum fructosamine
Reduction of glycosylated hemoglobin concentration
Attenuation of post-prandial glycemic response
Increase serum insulin concentration and reduce insulin resistance
169, 172, 173, 174, 177, 178, 179, 180, 181
170
171
176
170, 183, 186
Microbial infection Inhibition of microorganisms of clinical relevance 80, 212, 218
reported interesting results [111-117], in which flavonoids from
honey have also been studied [118-120].
Erythrocytes are the most abundant cells in the human body and
due to their structural and fun ction al characteristics they are targets
for continuous oxidative stress damage. Since oxidative damage to
the erythrocyte membrane is g enerally asso ciated with an increased
lipid peroxidation process, causing malfunctioning of the mem-
branes by altering its fluidity as well as the membrane-bound en-
zyme and receptor function, it has been proposed as a general
mechanism involved in cell injury and death, leading to erythrocyte
haemolysis [121-123]. In particular, in vitro tests have ascribed
antioxidant and antihemolytic properties of dietary flavonoids to
their localization in the membran e bilayer and to formation of se-
lective bindings with RBC membrane lipids and proteins, which
may exert a significant inhibition of lipid peroxidation, and enhance
membrane integrity against several chemical and physical stress
conditions [121, 122]. This mechanism appears to partially explain
how polyphenol extracts from honey were able to inhibit RBCs
oxidative hemolysis [118, 120], reduce the extracellular ferricya-
nide [119], protect against intracellular depletion of GSH and SOD
activity and to decrease the susceptibility of RBC lipid membrane
to peroxidation [118]. Another mechanism that may be involved in
RBC protection by honey flavonoids seems to be erythrocyte up-
take capacity of these molecules. Previous uptake studies in human
RBC showed an excellent incorporation of honey phenolics in RBC
[119]. Flavonoid uptake by RBCs and their interactions with mem-
brane were confirmed using quercetin as a reference standard model
because it has been widely identified in honey, is efficiently incor-
porated into erythrocytes, and finally, different studies have re-
ported its involvement in protecting RBCs membranes against oxi-
dative damage [71, 111, 113, 118, 120].
Epidemiological studies suggest that hypertension is a major
public health concern because of its high prevalence, besides also
being an important risk factor for the development of CVD, that
end-lasts in renal disease, stroke, and death [124, 125]. Research
has demonstrated a close relationship between oxidative stress and
hypertension, causing a vast interest in therapeutic approaches or
nutritional interventions to preventively decrease oxidative stress or
to treat hypertension itself [126, 127]. As a model for humans, Ere-
juwaet al .[128] evaluated the effect of honey supplementation on
elevated systolic blood pressure (SBP) in spontaneously hyperten-
sive rats (SHR) as well as the capacity of honey to ameliorate oxi-
dative stress in the kidney of SHR as a possible mechanism of its
antihypertensive effect. Their findings demonstrated that honey
supplementation significantly reduced SBP, and malondialdehyde
(MDA) levels in SHR. Recent studies indicate also that honey may
ameliorate susceptibility of kidney to oxidative stress in rats with
chronic renal failure or hypertension via up-regulation of Nrf2 ac-
tivity or expression [129]. In another study, the protection of honey
on the cardiovascular system was evaluated by Rakha et al. [130],
using a model of induction of hyperadrenergic activity in urethane-
anesthetized rats by epinephrine. Acute administration of epineph-
rine for 2 hours induced several cardiac disorders and vasomotor
dysfunction. The results showed that pretreatment with natural wild
honey (5 g/kg) for 1 hour prior to the injection significantly reduced
the incid ence o f epinephrine-induced cardiac disorders and vasomo-
tor dysfunction in anesthetized normal rats. Moreover, post-
treatment with natural wild honey, following the injection with
epinephrine for 1 hour, showed several ameliorative outcomes to
the electrocardiographic parameters and vasomotor dysfunction of
anesthetized stressed rats. From the results of this study it has been
hypothesized that honey may exert its cardioprotective effects
against epinephrine-induced cardiac disorders and vasomotor dys-
function directly, via its AOC and its great wealth of both enzy-
matic and non-enzymatic antioxidants involved in cardiovascular
defense mechanisms; also the contribution of substantial quantities
of mineral elements should be taken into account such as magne-
sium, sodium, and chlorine, and/or indirectly, the enhancement of
nitric oxide release, the endothelium-derived relaxing factor,
through the influence of ascorbic acid (vitamin C) [130].
Despite the results obtained using in vitro and animal models,
data from interventional studies in humans with CVD risk are few.
In a study of 55 overweight or obesepatients, supplementation of 70
g of natural honey against fructose-supplemented control for 30
days caused a mild reduction in body weight (1.3%) and body fat
(1.1%), a more consistent reduction of total cholesterol (3%), LDL-
C (5.8%), triacylglycerides (11%), and C-reactive protein (3.2%),
and an increase of HDL-C (3.3%) in subjects with normal values.
Meanwhile, in patients with impaired values, honey caused reduc-
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 9
tion in total cholesterol (3.3%), LDL-C (4.3%), triacylglycerides
(19%) and C-reactive protein (3.3%) [9]. Another study evaluated
the effects of the ingestion of 75 g of natural honey compared to the
same amount of artificial honey (fructose plus glucose) or glucose
in normal subjects and in both hypercholesterolemic and hyper-
triglyceridemic patients. Results showed that honey consumed for
15 days decreased cholesterol (7%), LDL-C (1%), TG (2%), C-
reactive protein (7%), homocysteine (6%), plasma glucose level
(6%), and increased HDL-C (2%) in normal subjects. In patients
with hypertriglyceridemia honey decreased TG while in subjects
with hyperlipidemia it decreased cholesterol (8%), LDL-C (11%),
and C-reactive protein (75%) after 15 days of consumption [131].
These results support the hypothesis that honey reduces cardiovas-
cular risk facto rs, particularly in subjects with elevated risk factors,
and it does not increase body weight in overweight or obese sub-
jects [9].
The consumption of honey could affect pathways related to
cardiovascular health by several mechanisms. The actions of poly-
phenols appear to be the main relevant mechanisms. The mains
flavonoids, quercetin and kaempferol, are reported as promising
pharmaceutical molecules in the treatment of cardiovascular dis-
eases, and may in some way help to understand the mechanisms by
which honey exerts its positive action ag ainst CVD.Quercetin is
rapidly conjugated with glucuronic acid and/ or sulfate during a
first metabolism step and a portion of the metabolites are also
methylated, reaching the blood stream as methylated, glucuroni-
dated and sulfated products [132]: for example, it has been recently
reported that glucuronidated and sulfated metabolites of quercetin
exhibited a protective effect on endothelial dysfunction [133]. In
cultured human endothelial cells quercetin has been shown to both
up-regulate eNOS gene expression [134] and stimulate the
NO/cGMP pathway [135, 136]: these findings are of utmost interest
since currently the endothelial NOS gene is a candidate in investi-
gations on CVD genetics by the established role of NO in vascular
homeostasis. Other studies in vitro and in animal models have also
shown that quercetin can increase the activity of eNOS and stimu-
late arterial relaxation [137], possibly via activation of Ca2+-
activated K+ channels [138]. Moreover, Shen et al.[139] reported
that quercetin and its major in vivo metabolites can protect vessels
against hypochlorous acid-induced endothelial dysfunction in iso-
lated arteries, presumably mediated in part, by an adenosine mono-
phosphate-activated protein kinase (AMPK) pathway. AMPK acti-
vation leads to subsequent eNOS activation and increased NO pro-
duction, suggesting that beneficial effects of quercetin on endothe-
lial cell functions are in part mediated via AMPK pathway.
In animal studies it was found that chronic treatment with die-
tary quercetin lowers blood pressure and restores endothelial dys-
function in hypertensive animal models. In a model using sponta-
neously hypertensive rats (SHR) (5 weeks old), animals were
treated with quercetin (10 mg/kg) for 13 weeks. In these animals
quercetin reduced blood pressure and heart rate, and enhanced the
endothelium-dependent aortic vasodilation induced by acetylcho-
line. These findings suggest that enhanced eNOS activity, and de-
creased NADPH oxidase-mediated superoxide anion generation
associated with reduced p47 expression appear to be the essential
mechanisms for the improvement of endothelial function and the
antihypertensive effects of chronic quercetin intake [139]. In a re-
cent research Panchal et al. [140] reported that rats supplemented
with quercetin presented a higher protein expression of nuclear
factor (erythroid-derived 2)-related factor-2 (Nrf2), heme oxy-
genase-1, and carnitine palmitoyltransferase 1 and lower expression
of NF-B, compared with the control group. Moreover, the sup-
plemented animals showed less abdominal fat and lower systolic
blood pressure along with attenuation of changes in structure and
function of the heart and liver compared with control rats. Thereby,
quercetin treatment attenuated most of the symptoms of metabolic
syndrome, including abdominal obesity, cardiovascular remodeling,
with the most likely mechanisms being the decrease in oxidative
stress and inflammation. These data are in acco rdance also with the
results published by Jung et al. [141] where quercetin reduces fat
accumulation in C57B1/6 the liver of mice that have undergone a
high-fat diet, due to its capacity to regulate the lipogenesis at tran-
scription level.
Another common flavonoid in honey with a putative important
role in the treatment of cardiovascular diseases is kaempferol. Some
researchers have elucidated a group of mechanisms by which
kaempferol exhibits its beneficial properties to the cardiovascular
system. The protective effect of kaempferol against endothelial
damage seems to be correlated with its ability to improve NO pro-
duction and to decrease asymmetric dimethylarginine (ADMA)
levels. Experiments have been performed using aorta and plasma
from C57BL/6J control and apolipoprotein E-deficient (ApoE/)
mice treated or not with kaempferol (50 or 100 mg/kg, intragastri-
cally) for 4 weeks, as well as human umbilical vein endothelial
cells (HUVECs) pretreated or not with kaempferol (1, 3 or 10 μM)
for 1 h and then exposed to lysophosphatidylcholine (LPC) (10
μg/mL) for 24 h [142]. Treatment with kaempferol improved endo-
thelium-dependent vasorelaxation, increased the maximal relaxa-
tion valu e, and decreased its half-maximum effective concentration
concomitantly with an increase in nitric oxide plasma concentra-
tion, and a decrease in ADMA and MDA plasma concentrations.
Moreover, these compounds caused an increase in the expression of
aortic endothelial NOS as well as of dimethylarginine dimethy-
laminohydrolase II (DDAH II) in ApoE/ mice. Finally, it was
found that kaempferol abolished the reduction of NO production,
the increase in ADMA concentration and the decreased expression
of eNOS and DDAH II in HUVECs caused by LPC [142]. In an in
vitro study using isolated porcine coronary artery rings the vascular
effects of kaempferol were studied by Xu et al.[143]. The results
demonstrated that kaempferol enhanced the relaxation produced by
bradykinin, isoproterenol and sodium nitroprusside in endothelium-
intact porcine coronary arteries. It was concluded that kaempferol
in a dose d ependent mod e, has the ability to enhance endothelium-
dependent and endothelium-independent relaxations, which is not
related w ith its antioxidant properties.
Besides its aglycon form, kaempferol has demonstrated protect-
ing effects on CVD also in its glycosylated form. It was also dem-
onstrated that kaempferol-3-O-sophoroside (KPOS) inhibited LPS-
induced barrier disruption, expression of cell adhesion molecules,
neutrophil adhesion and trans-endothelial migration of neutrophils
to human umbilical vein endothelial cells (HUVECs). Further stud-
ies revealed that KPOS suppressed the production of TNF- and the
activation of NF-B by lipopolysaccharides. These results suggest
that KPOS possesses barrier integrity activity, inhibitory activity on
cell adhesion and migration to endothelial cells by blocking the
activation of NF -B expression and production of TNF-, thereby
endorsing its usefulness as therapy for vascular inflammatory dis-
eases [144]. Although these studies provide new insights on the
potential functional benefits of honey antioxidant compounds, fur-
ther investigations in animal and human mod els are need ed to con-
firm the hypothesized vascular protective effects of honey.
4.2 Honey and Cancer
Currently, there are few studies reporting the effect of honey in
cancer, although interest in this area is growing among researchers.
A group of studies has been particularly focused on the efficacy of
crude honey or its components in inhibiting mutagenesis or induc-
ing apoptosis, and the transformation of different types cancer cell
and proliferation in vitro; moreover, the mechanisms underlying the
anti-tumorigenic effects of honey at the cellular and molecular lev-
els are still limited. The major components of honey, i.e., sugar,
particularly glucose and fructose, have been reported to display
both mutagenic and antimutagenic effects in different systems;
antioxidants, in their turn, often show antimutagenic activity [145].
10 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
As a result, since honey is a rich source of dietary sugars, little is
known about possible actual antimutagenic effects. In an in vitro
model Wang et al.[145] studied the antimutagenic capacity of hon-
eys from seven different floral origins against the encountered food
mutagen Trp-p-1, comparing it to that of a sugar analogue and to
individually tested simple sugars. The results showed that all hon-
eys studied exhibited significant inhibition against Trp-p-1
mutagenicity. In teresting was the fact that sugars selected for analy-
sis, either individually or in combination demonstrated a pattern of
inhibition similar to that of honeys, where glucose and fructose
were also similar to honeys and were more antimutagenic than mal-
tose and sucrose. From these results it may be assumed that honey
polyphenols are not solely responsibles for honey antimutagenic
activity. It is known that sugars can display both mutagenic and
antimutagenic effects in different systems [146], and since honey is
a rich mixture of sugars, its use as a factor able to prevent mutage-
nesiscould result interesting.
The induction of apoptosis by honey is another capacity that has
been recently highlighted. Jaganathan et al., [147] tested the apop-
totic effect of selected crude honey samples in colon cancer cell
lines (HCT 15 and HT 29). Pretreatment of cells with honey pro-
duced a significant dose-dependent anti-proliferative effect, show-
ing the increasing accumulation of hypodiploid nuclei in the sub-G1
phase of cell cycle that indicated apoptosis. In this cell model the
same authors also reported that honey transduced the apoptotic
signal via initial depletion of intracellular non protein thiols, conse-
quently reducing the mitochondrial membrane potential and in-
creasing reactive oxygen species generation. Honey induced apop-
tosis was accompanied by up-regulating p53 and modulating the
expression of pro and anti-apoptotic proteins. Further, in HT 29
cells, honey elevated caspase-3 level and displayed typical ladder
pattern, confirming apoptosis [147]. Another study tested the anti-
proliferative role of acacia honey and chrysin, as a major natural
flavone found in this honey in human (A375) and murine (B16-F1)
melanoma cell lines. The results showed that both compounds were
able to induce an antiproliferative effect on melanoma cells in a
dose- and time-dependent manner, mediating by G(0)/G(1) cell
cycle arrest and induction of hyperploid progression [12]. Similarly,
Tualang honey was investigated using human breast (MCF-7 and
MDA-MB-231) and cervical (HeLa) cancer cell lines, as well as
normal breast epithelial cell line (MCF-10A). After 72 h of incuba-
tion with increasing doses of honey (1-10%) an increase in lactate
dehydrogenase (LDH) leakage from the cell membranes was evi-
denced, indicating a cytotoxic effect of honey to all the three cancer
cell lines, while no cytotoxic effects was evidence in MCF-10A
cell. Honey also reduced the mitochondrial membrane potential
((m)) in cancer cell lines after 24h of treatment. Moreover, the
activation of caspase-3, -7 and -9 was observed in all honey-treated
cancer cells indicating the involvement of mitochondrial apoptotic
pathway [148]. Tualang honey was also tested in oral squamous cell
carcinomas (OSCC) and human osteosarcoma cells (HOS) [149]. In
this study, the results in morphological appearance showed signifi-
cant apoptotic cellular changes in both treated cell lines (rounded,
reduction in cell number, blebbed membrane), as well as significa-
tive apoptotic nuclear changes (nuclear shrinkage, chromatin con-
densation and fragmented nucleu s). Moreover, cell viability assay
showed a time and dose-dependent inhibitory effect on both cell
lines, where signals of early apoptosis were evident, with the per-
centage of early apoptotic cells which increased in a dose and time
dependent manner [149].
Despite a consistentamount of results obtained using in vitro
models, data from in vivo studies are still very limited. In bladder
cancer cells, the antitumor effect of honey was examined both in
vitro and in vivo. Three human bladder cancer cell (T24, 253J and
RT4) and one murine bladder cancer cell lines (MBT-2) were used.
The in vitro studies revealed significant inhibition of the prolifera-
tion of T24 and MBT-2 cell lines and of RT4 and 253J cell lines. A
lower S-phase fractionwas also found, as well as absence of ane-
uploidy compared with control cells. Moreover, in the in vivo stud-
ies, researchers observed that intralesional injection of 6 and 12%
honey and oral ingestion of honey significantly inhibited tumor
growth [150]. Honey also exerted a pronounced antimetastatic ef-
fect in murine tumor models (mammary carcinoma (MC a) and a
methylcholanth rene-induced fibrosarcoma (FS)) when administered
before tumor cell inoculation (2 g/kg orally once a day for 10 con-
secutive days) [151]. The results presented by Attia et al. [152]
have shown other possible routes by which honey can exert its anti-
tumor capacity (EAT). In this study the antitumor effect of honey
against EAC in mice and the possible mode of its antitumor action
were investigated. Results evidenced that pre-oral administration of
mice with different honey doses before intraperitoneal inoculation
with EAT (1 x 106 cells) increased the number of bone marrow
cells as well as peritoneal macrophages, but not peripheral blood
leukocytes. An increase in phagocytic functions of macrophages-
was also found as well as T- and B-cell functions. Moreover, in
vitro studies on EAT cells demonstrated an inhibitory effect of
honey on tumor cell proliferation, on the viability % of tumor cells
as well as on the size of solid tumor. These results allow to hy-
pothesize that honey antitumor activity may also occur via activa-
tion of macrophages, T- and B-cells [152].
Honey constituents, as polyphenols, have shown complemen-
tary and overlapping mechanisms of chemopreventive activity in
multistage carcinogenesis [17]. Dietary flavonoids may be used as
chemotherapeutics and preventatives against critical health condi-
tions such as cancer. The large number of effects of flavonoids, like
major polyphenolics in honey, on the metabolism of cancer cells is
difficult to summarize in a few b asic and specific mechanisms.
Several biochemical structures and pathways related to carcino-
genesis can be influenced by flavonoids like: (i) cytoplas-
mic/nuclear hormone receptors, as the most highly sensitive to
flavonoids; (ii) enzymatic, by inhibition of several enzymes in-
volved in the oncogenesis, while the reactions of some phospha-
tases and oxygenases are improved; (iii) growth regulation, by
inhibition of pathways for the transmission of environmental sig-
nals to the genes as the steroid path via the cytoplasmic receptor
and the protein kinase cascades; (iv) energy metabolism, by inhibi-
tion of glycolysis which leads to a depletion of ATP, especially in
tumor cells with mitochondrial respiratory defects. These processes
lead to a rapid dephosphorylation of the BAD molecules integrative
of glycolysis-apoptosis in SER, a relocation of BAX to mitochon-
dria and massive cell death, possibly due to removal of the inhibit-
ing phosphate residue on the -chain of the Na+/K+ pump
[17].These effects were attributed to two fundamental properties of
flavonoids: the electronic and the steric characteristics. The first
one is due to the h igh mobility of the electrons in the benzenoid
nucleus of flavonoids which accounts for both their antioxidant and
free-radical scavenging properties; the second one is due to the
structural similarity between the aglycone form of flavonoids and
various compounds involved in normal cell biochemistry such as
nucleic acid bases, coenzymes, steroid hormones, neurotransmit-
ters, cytoplasmic/nuclear hormone receptors, as well as gene induc-
tion. Moreover, the high affinity of flavonoids for heavy metal ions
allows to interfere with the action of enzymes and Zn2+ fingers in
DNA-binding proteins [17].
Quercetin has been the subject of several studies that have
shown a significant antiproliferative activity in different tumor
lines, and since it is one of the mostfrequentlyflavonoids identified
in honey, its presence could help, in part, to explain honey antipro-
liferative properties. Since it is rapidly absorbed and metabolized in
circulating cells, its effects in several in vitro models of leukemia
focused the attention of researchers. Kang et al. [153] investigated
the role of quercetin in human promyelocytic leukemia cells (HL-
60). Pretreatment of HL-60 cells with quercetin produced a dose-
dependent inhibition on the activities of cytosolic protein kinase C
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 11
(PKC) and two pore K+ (TPK). Cell cycle analysis indicated that
quercetin suppressed in a dose-dependent mode the number of cells
in the G2/M phase and decreased the population of G0/G1 cells. It
was also found that quercetin repressed the complete activity of
phosphoinositides like phosphatidylinositol (PI), phosphatidylinosi-
tol 4-phosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate
(PIP2): it could be concluded that the inhibitory effect of quercetin
on the growth of HL-60 cells may be related to its inhibitory effects
on PKC and/or TPK in vitro and/or on the production of phosphoi-
nositides. More recently, it was been reported that quercetin also
induced fast ligand–related apoptosis in these cells by promotion of
histone H3 acetylation [154]. In other cellular models, such as
chronic myeloid leukemia (CML) cells, quercetin caused a deple-
tion of the activity of telomerase [155], a decrease of the level of
Notch1 protein [156] and inhibition of murine leukemia WEHI-3
cells when injected into BALB/c mice, as well as a promotion of
macrophage phagocytosis and natural killer cell activity [157].
Studies conducted by Russo et al. [158] in B cells isolated from
chronic lymphocytic patients (B-CLL) reported that quercetin en-
hanced sensitiv ity to anti-CD95 and rTRAIL treatment with an
increase in cell death of about 1.5- and 1.6-fold, respectively, when
compared with quercetin monotreatment, suggesting that quercetin
supplementation may have the capacity to strengthen the efficacy of
drugs widely used in the therapy of B-CLL, such as fludarabine
[159].
The antitumor effects of quercetin are also reported in a large
number of tumor cell models. Recent studies in breast cancer cell
(MDA-MB-435, MCF-7) showed an inhibitory effect on cell
growth in a time and dose dependent manner. The analysis of cell
cycle in quercetin treated cells showed significant increase in the
accumulation of cells at subG1 phase. Quercetin treatment also
increased Bax expression but decreased the Bcl2 levels, while
cleaved caspase-3 and PARP expression were increased [159, 160].
In human glioma cell cultures (U138MG) Braganhol et al. [161]
reported that qu ercetin decreased cell proliferation and viability b y
necrotic and apoptotic cell death, arrest the G2 checkpoint of the
cell cycle, and decreased the mitotic index. Furthermore, it was also
found that quercetin was able to protect the hippocampal organo-
typic cultures from ischemic damag e. Therefore, all th ese results
allowed to hypothesize that the main routes by which quercetin is
capable of regulating tumor cell growth are focused on its ability to
induce apoptosis by decreasing the Bcl2 levels, increasing Bax
expression, cleaved caspase-3 and PARP expression, stop cell cy-
cle, and arrest the cell cycle in the G2/M phase, proving it to be an
active compound with potential uses like chemotherapeutics and
preventatives supplied by diet.
Besides quercetin, other flavonoids identified in honey such as
kaempferol, are responsible for significant antiproliferative effects.
Using the HL-60 cells, it was found that kaempferol caused cell
cycle alteration s, with a significant increase of cells in S-phase and
a progressive accumulation in G2/M, while cells with apoptotic
indices confirmed a heightened caspase-3 activity and a decreasing
of anti-apoptotic Bcl-2 expression [162]. Kaempferol also showed
the capacity of reducing the risk of ovarian cancer. Recently, Luo et
al.[163] demonstrated that kaempferol in a time-dependently way
inhibited vascular endothelial growth factor (VEGF) secretion, and
suppressed in vit ro angiogenesis in ovarian cancer cells.
Kaempferol also down-regulated extracellular signal-regulated
kinases (ERK) phosphorylation as well as NFB and cMyc expres-
sion, but promoted p21 expression, suggesting a novel ERK-NFB-
cMyc-p21-VEGF pathway, which accounts for kaempferol angio-
prevention effects in ovarian cancer cells.
Among honey phytochemicals, phenolic acids like caffeic acid
and its esters have also been associated to the chemopreventive
effects of honey, appearing to function as an anticarcinogen at the
initiation and post-initiation stages of tumor development in in vitro
and in vivo experiments [164, 165]. The studies using HCT 15 and
HT 29 colon cancer cells demonstrated that caffeic acid signifi-
cantly inhibited the cell proliferation. The cell-cycle analysis in
caffeic acid-treated cells indicated increasing accumulation of cells
at sub-G1 phase, accompanied by an increasing ROS generation
and reduction in the mitochondrial membrane potential, confirming
a dose- and time-dependent apoptotic effect of caffeic acid [164-
166]. Moreover, when caffeic acid phenethyl ester (CAPE) was
explored on growth, cell cycle, apoptosis and beta-catenin/T-cell
factor signaling in human colon cancer cell (HCT116 and SW480)
it completely inhibited growth, and induced G1 phase arrest and
apoptosis in a dose-dependent manner. In treated cells a dose-
dependent and time-dependent loss of total beta-Catenin proteinwas
also found, associated with a decrease of nuclear beta-catenin, as
well as a reduction of the expression of cyclin D1 and c-myc [167].
The studies in other tumo r cell lines also confirmed the antiprolif-
erative effect of caffeic acid. A research in human cervical cancer
cells (HeLa cells) conducted by Chang et al. [168] showed that
caffeic acid significantly induces apoptosis in HeLa cells in a con-
centration-dependent manner by inhibiting Bcl-2 activity, leading to
release cytochrome c and subsequent activation of caspase-3 and
p53. These results indicate that caffeic acid and its esters are excel-
lent inducers of apoptosis in tumor cell lines, allowing to hypothe-
size about the other possible mechanisms by which honey exerts its
antitumor effects.
Results in vitro and in vivo are encouraging and demonstrate the
potential uses of honey as a possible preventive agent against the
development of degenerative diseases. Certainly, other potential
mechanisms of honey anticancer activities, already investigated
with others polyphenolic rich-food, remain to be evaluated, such as
the ability to interact/interfering with an environmental carcino-
genic uptake or activation, or the capacity of inhibiting matrix met-
alloproteinases and other enzyme families implicated in cancer
metastasis, just to mention some of them .
4.3 Honey and Diabetes
Honey can positively affect the glycemic response by reducing
blood glucose [169], serum fructosamine [170] or glycosylated
hemoglobin concentration [171]. Animal studies demonstrated that
honey supplementation significantly decreases glycemic values in
both diabetic and non-diabetic rabbits [172], and it reduces blood
glucose concentrations in alloxan-induced [169] and in streptozoto-
cin-induced (STZ-induced) diabetic rats in a dose-dependent mode
[173, 174]. The data referring to the effects of honey on fructosa-
mine or glycosylated hemoglobin levels are still limited; however,
it has been reported that chronic honey supplementation reduces
glycosylated hemoglobin in non-diabetic rats [171], while in STZ-
induced diabet ic rats it decreases significantly serum concentrations
of fructosamine [170]. Moreover, the combination of antidiabetic
drugs, such as glibenclamide or metformin, with honey results in
further reductions in serum concentrations of both glucose and fruc-
tosamine in STZ-induced diabetic rats [170].
Evidence from clinical studies showed that honey, compared
with dextrose, sucrose or other sweeteners, attenuated post-prandial
glycemic response in non-diabetic volunteers [175]. In healthy hu-
man subjects, it was found that natural honey consumption (1g/kg
body weight) decreases glycemic post-prandial response compared
to artificial honey and D-glucose which elevated blood glucose
levels by 47% and 52%, respectively after 60 minutes [176]. In
patients with diabetes mellitus, honey supplementation significantly
reduced postprandial glycemic response causing a lower rise in
plasma glucose compared with other sugars or sweeteners [176].
Similarly, honey decreased the concentrations of blood glucose in
patients with type 2 diabetes mellitus [177,178]. Compared to su-
crose, honey lowered glycemic and peak incremental indices in
type 1 diabetic patients [179], while in children with type 1 diabetes
mellitus honey reduced hyperglycemia [180]. A glucose-lowering
effect of honey was also reported in subjects with impaired glucose
12 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
tolerance [181]. In this study subjects presented significantly lower
plasma glucose concentrations after consumption of honey at all-
time points of the honey tolerance test in comparison to the oral
glucose tolerance test. Plasma glucose levels in response to honey
peaked at 30-60 minutes and showed a rapid decline, when com-
pared to glucose. Significantly, the high degree of tolerance to
honey was recorded in subjects with diabetes, suggesting a lower
glycemic index for honey. However, despite the evidence of honey
hypoglycemic effect, some studies found no beneficial effect of
honey on hyperglycemia in type 2 diabetic patients and non-
diabetic rats [182, 183], possibly caused by the short duration of
honey supplementation or feeding [184].
From the above mentioned articles, it can be evinced that honey
consumption or its addition to dietary carbohydrates could be bene-
ficial in individuals with diabetes. However, few studies have stud-
ied fructosamine or glycosylated hemoglobin in diabetic patients
after honey supplementation, making it difficult to ascertain the
actual effect of honey on these parameters in diabetic patients. The
antidiabetic mechanisms by which honey exerts its glycemic con-
trol have also been associated with its capacity to modulate key
glucose-regulating hormones, especially insulin [185]. Studies in
healthy subjects demonstrated that honey supplementation, com-
pared with glucose or the combination of glucose-fructose solution,
produced significantly lower serum insulin and C-peptide concen-
trations [186]. In diabetic patients, honey supplementation in-
creased insulin concentrations more than sucrose [186], while in
type 2 diabetics reduced insulin resistance [183]. The effect of
honey on glucose-regulating hormones and pancreas was also re-
ported in animal studies using STZ-induced diabetic rats, where
honey supplementation was associated with a considerable im-
provement in pancreatic islets as well as increased serum insulin
levels [170].
Fructose and glucose, the main sugars present in honey, are in-
volved in some mechanisms related to the glycemic control. Studies
either in diabetic rodent models or healthy and diabetic subjects
have shown that fructose reduces hyperglycemia [187-190]. Evi-
dence suggests that fructose contributes in regulating blood sugar
levels by slowing digestion [191], prolonging gastric emptying and
slowing down the rate of intestinal absorption [192]. Besides fruc-
tose, glucose is the second major sugar constituent in honey [7]. A
significant synergy has been reported between these molecules
which actively influence their absorption, indicating that intestinal
absorption of fructose is improved in the presence of glucose [193].
Fructose and glucose have different transporters, GLUT5 (and/or
GLUT2) and SGLT1, respectively [194]; despite this, actually the
mechanism by which glucose enhances fructose absorption remains
unclear. The recruitment of GLUT2 carrier to the brush border
membrane caused by increased intestinal fructose may contribute to
the synergistic effect of glucose on the absorption of fructose [193].
The general schema of the absorption of glucose and fructose by
enterocytes in the sm all intestine is shown in Fig. (5). SGLT1 is
expressed in the brush border membrane of the enterocyte, where it
couples the transport of two sodium ions and one glucose molecule
across the brush border membrane. The energy produced by the
sodium electrochemical potential gradient across the brush border
membrane is used to facilitate the glucose accumulation inside of
enterocyte against its concentration gradient. The sodium ion that
enters the cell along with glucose is then transported out into blood
through Na/K-pump in the basolateral membrane, allowing to main-
tain the driving force for glucose transport. The accumulation of
sugar into enterocytes generates a driving force to transport glucose
from cells into the blood via GLUT2, expressed in the basolateral
membranes of enterocyte. A fraction of the intracellular glucose
seems to be taken up into endosomes, as glucose-6-phosphate, and
then released into the blood by exocytosis through the basolateral
membrane [195]. Otherwise, after ingestion of fructose, unlike glu-
cose, an increase in the expression levels of GLUT5 mRNA was
found [196]. It was also suggested that there may be a disacchari-
dase-related transport system which considers both fructose and
glucose production from the enzymatic hydrolysis of sucrose [197].
Other evidence suggests that fructose is absorbed via a saturable
carrier in the ab sence o f glucose, wh ile in th e presence of gluco se;
fructose is absorbed via a disaccharidase-related transport system
[197]. Besides this, passive diffusion across the intestinal epithe-
lium has also been proposed as a possible mechanism [197]. Studies
Fig. (5). Mechanisms for the absorption of glucose and fructose in the small intestine. F, fructose; G, glucose; GLUT2, glucose transporter; GLUT5, fructose
transporter; G6P, glucose-6-phosphate; SGLT1, sodium-dependent glucose transporter.
Honey and Human Health Current Medicinal Chemistry, 2013, Vol. 20, No. 1 13
have shown that glucose improves the transportation and absorption
of fructose but not vice versa, increasing the amounts of fructose
that reach the liver. According to the results of the investig ations
exposed above, the potential role of honey against diabetes mellitus
is at an early stage, where more specific research es are needed to
understand the mechanisms by which it can exert its hypoglycemic
action. Even if these studies are still scarce, they have shown that
honey is preferable to the most common sugars or sweeteners, be-
cause it is more tolerable both in healthy subjects and in patients
with diabetes mellitus. Moreover, its consumption or its addition to
other carbohydrates can be recommended in diabetic patients, be-
cause of its minimal incremental effect on blood glucose compared
to other sweeteners or common sugars.
4.4 Antimicrobial Action of Honey
The antimicrobial activity of honeycan be divided into two fun-
damental mechanisms: (i) a non-peroxide antibacterial activity,
mainly due to its high osmolarity and acidity as well as to methyl-
glyoxal, bee defensing-1and flavonoid contents; (ii) a peroxide-
associated antibacterial activity due to the specific hydrogen perox-
ide content [30, 198, 199].
The inhibition of microorganisms of clinical significance car-
ried out by honey has been widely reported in many studies. Cooper
et al. (2002) well established the antimicrobial activity of manuka
and pasture honeys and made a comparison with an artificial honey
solution using eighteen strains of methicillin-resistant Staphylococ-
cus aureus, seven strains of vancomycin-sensitive enterococci,
isolated from infected wounds, and 20 strains of vancomycin-
resistant enterococci, isolated from hospital environmental surfaces.
The authors reported that for all of the strains tested, the minimum
inhibitory concentration of both honeys was below 10%, while the
concentrations of artificial honey necessary to achieve equivalent
inhibition in vitro were at least three times higher, thus confirming
that the inhibition of bacteria by honey is not exclusively due to its
osmolarity. In another study, the antimicrobial activity of five na-
tive monofloral Cuban honeys against four bacterial strains, two
gram-positive (Bacillus subtilis, ATCC 6633 and Staphylococcus
aureus, ATCC 25923) and two gram negative species (Pseudo-
monas aeruginosa, ATCC 27853 and Escherichia coli, AT CC
25922), was reported [80]. The results indicated that S. aureus was
the most sensitive microorganism, while P. aeruginosa was the
most resistant. Moreov er, B subtilis and E. coli were moderately
sensitive to the antimicrobial activity of honey. In general, the re-
sults of this study showed that Gram-positive bacteria were more
sensitive to the honey antimicrobial action than Gram-negative
bacteria. Other authors have also reported S. aureus as the most
sensitive microorganism to honey antimicrobial action:since S.
aureus has been reported as the causal agent of a range of illnesses
from skin infections to life threatening diseases, such as pneumonia
and meningitis, this is an important achievement that suggest to
consider honey as a possible treatment against this agent [200-209].
Honey also presents inhibitory activity against Pseudomoma aeru-
ginosa, Bacillus anthracis (anthrax), Corynebacterium diphtherine
(diphtheria), Klebsiella pneumoniae (pneumonia), Mycobacterim
tuberculosis (tuberculosis), Salmonella typhi (typhoid fever), Vib ri o
cholerae (cholera) [210].
As indicated above, the antibacterial activity of honey is due to
the involvement of multiple compounds and to the contribution of
individual components to its total antibacterial activity. Recently
researchers have focused their attention on the presence of methyl-
glyoxal, a component that contributes to honey non-peroxide anti-
bacterial activity: methylglyoxal, (CH3-CO-CH=O) is the aldehyde
form of pyruvic acid formed by two carbonyl groups (Fig. 6). This
compound is formed from sugars during heat treatment or pro-
longed storage of carbohydrate-containing foods and beverages
[211]. High levels of methylglyoxal have been found in manuka
honey [212, 213], one of the components mainly responsible for its
non-peroxide antibacterial activity [213]. Its high concentration in
manuka honey is due to the conversion of dihydroxyacetone, which
is present in great amounts in the nectar ofL. scoparium flowers
[214], and it occurs non-enzymatically at a slow rate during storage
of honey. The study demonstrated a strong correlation between
methylglyoxal levels and the potential of honey to inhibit the
growth of S. aureus. In a different research, it was suggested that
methylglyoxal may be fully responsible also for the non-peroxide
antibacterial activity of manuka honey [212].
Fig. (6). Chemical structures of the methylglyoxal.
The amino- and carboxy-termini are labelled N and C, respec-
tively
Another antibacterial component in honey is bee defensin-1
peptide (Fig. 7). This peptide has been previously identified in hon-
eybee hemolymph [215], honeybee head and thoracic glands [216]
and in royal jelly [217], and a potent activity was reported against
Gram-positive bacteria including B. subtilis, S. aureus, and Paeni-
bacillus larvae [218]. Kwakman et al. [218] are the authors of the
first report of this peptide in honey and they also investigated its
antibacterial capacity in their recent analysis of unprocessed Re-
vamil honey. Despite this interesting finding, the presence of bee
defensin-1 in honeys has not been investigated systematically, and
quantitative data on the concentration of this peptide in honey have
not yet been established.
Fig. (7). Homology model of defensin-1 from Apis mellifera. The model of
the mature protein (residues 44-94) was obtained using the experimentally
resolved structure of lucifensin from Lucilia sericata (PDB ID: 2LLD) as a
template. Alig nment and modelling was performed using the SwissModel
server (http://swissmodel.expasy.org). Disulfide bridges are shown as sticks.
Finally, the non-peroxide antibacterial activity of honey and its
relationship with its own flavonoid composition have also been
partially ascertained. Several antibacterial phenolic compounds
have been identified in honeys [219-221], but their contribution to
the antimicrobial activity of honey remains unclear. The use of
flavonoids against bacterial infections has two purposes: (i) to kill
the bacterial cells and (ii) to counteract the spread and the effects of
the bacterial toxins [17]. The bactericidal effect of flavonoids ap-
pears to be the result of a metabolic perturbation related with io n
channels, which are especially sensitive points of inhibition and
likely targets of flavonoids [17]. Besides the active role that the
flavonoids play in the destruction of infectants, they fortify loose
connective tissues by inhibiting some of the enzymes that can hy-
14 Current Medicinal Chemistry, 2013, Vol. 20, No. 1 Alvarez-Suarez et al.
drolyze their proteoglycan and protein meshwork, making the diffu-
sion of infections through the tissue sterically difficult [17].
The incomplete knowledge of antibacterial compounds in-
volved in the antibacterial activity of honey is an obstacle for wide
clinical use of honey. In recent years, the knowledge on the antibac-
terial compounds in honey markedly increased. The results on
honey antimicrobial properties are encouraging and demonstrate the
potential uses of honey as an antibacterial agent, where its powerful
activity against antibiotic-resistant bacteria could be an effective
mode to counteract these agents.
CONCLUSIONS
Honeys are a natural source of phytochemical compounds
mostly represented by polyphenols. The evidence of the biological
action correlated to their polyphenolic content has been demon-
strated. Honey compounds have been associated to antioxidant and
anti-inflammatory actions, reporting cardiovascular, antiprolifera-
tive, and antimicrobial benefits. Although most health-promoting
effects were initially observed with in vitro studies, there are in-
creasing animal and clinical researches fo cused on tran slating the in
vitro evid ence into in vivo outcomes. A greater understanding of the
mechanisms and factors governing the bioavailability of honey
phytochemicals will be crucial to understanding the mechanisms by
which honey exerts its beneficial effects on human health.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts
of interest.
ACKNOLEDGEMENTS
The authors wish to thank the Università Politecnica delle
Marche, Ancona, Italy for supporting this work and Ms. Monica
Glebocki for extensively editing the manuscript.
ABBREVIATIONS
AAPH = 2,2´-azobis(2-methylpropionamidine dihydro-
chloride
ADMA = Dimethylarginine
AMPK = Adenosine monophosphate-activated protein
kinase
AOC = Antioxidant capacity
ApoE/ = Apolipoprotein E-deficient mice
Bcl2 = B-cell lymphoma 2
CBG = Cytosolic -glucosidase
COMTs = Catechol-O'M ethyltransferase
CVD = Cardiovascular diseases
DDAH II = Dimethylarginine dimethylaminohydrolase II
EAT = Ehrlich ascites tumor
eNOS = Endothelial nitric oxide synthase
ERK = Extracellular signal-regulated kinases
GLUT2 = Glucose transporter
GLUT5 = Glucose transporter
GOx = Glucose oxidase
HDL-C = High-density lipoprotein
HeLa cells = Human cervical cancer cells
HL-60 = Human promyelocytic leukemia cells
HUVECs = Human umbilical vein endothelial cells
KPOS = Kaempferol-3-O-sophoroside
LDL = Low-density lipoprotein
LDL-C = Low-density lipoprotein
LPC = Lysophosphatidylcholine
LPH = Lactase phlorizin hydrolase
MDA = Malondialdehyde
MRP-1 = Multidrug-resistance-associated proteins
MRP-2 = Multidrug-resistance-associated proteins
MRP-3 = Multidrug-resistance-associated proteins
NF-B = Nuclear factor kappa-light-chain-enhancer of
activated B cell
NO = Nitric oxide
Nrf2 = Nuclear factor (ery throid-derived 2)-related
factor-2
OH = Hydroxyl group
PARP = Poly (ADP-ribose) polymerase
PKC = Cytosolic protein kinase C
RBC = Red blood cells
RDI = Recommended daily intake of energy
RNS = Reactive nitrogen species
ROS = Reactive oxygen species
SBP = Systolic blood pressure
SGLT1 = Sodium-dependent glucose transporter 1
SHR = Spontaneously hypertensive rats
SHR = Spontaneously hypertensive rats
SOD = Superoxide dismutase
STZ-induced = Streptozotocin-induced
SULTs = Sulfotransferases
TG = Triacylglycerid es
TNF- = Tumor necrosis factor-alpha
TPK = Two pore K+
Trp-P-1 = 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole
UGT = Uridine-5`-diphosphate glucuronosyltrans-
ferases
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