How do bees prevent hive infections? The antimicrobial properties of
, A. Pereira
, A. M. Ferreira
, A. Cunha
, and C. Aguiar
Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Department of Chemistry, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
CQVR (Chemistry Centre of Vila Real), 5001-801 Vila Real, Portugal
CITAB (Centre for the Research and Technology of Agro-Environmental and Biological Sciences), Campus de Gualtar,
4710-057 Braga, Portugal
CBMA (Molecular and Environmental Biology Centre), Campus de Gualtar, 4710-057 Braga, Portugal
Propolis is a wax-like resin produced by honeybees from substances collected from plants, which are mixed with beeswax
and other compounds of bee metabolism. Its chemical composition depends on the specific local flora at the site of
collection and also of climatic characteristics, resulting in a striking diversity of constituents. Propolis is a mixture of
balsams and resins, waxes, essential oils, pollen, and other substances which is used by bees in the construction, repair and
protection of their hives, mainly due to its mechanical properties and antimicrobial activity. Because of the broad spectrum
of biological activities and medicinal properties, propolis has been used by man since ancient times. Nowadays it is still
used in traditional and alternative medicine, but also in the modern biocosmetic industry and in health foods. The renewed
interest in this natural product is due to its antimicrobial, anticancer, antioxidant, antiviral, and other properties. Here we
review the current knowledge about propolis diversity (geographic, compositional), its biological activities with emphasis
to antimicrobial activity, and its potential therapeutic applications. Propolis ecological functions are also discussed.
Keywords propolis, bioactivities, antimicrobial
Propolis or bee glue, as it is commonly named, is a natural resinous mixture produced by honeybees (Apis mellifera)
from substances collected from parts of plants, buds and exudates . This resin is masticated, salivary enzymes are
added, and then it is mixed with beeswax and probably with other compounds of bee metabolism . Etymologically
the word propolis derives from the Greek pro (for ‘in front of’, ‘at the entrance to’) and polis (for ‘community’ or
‘city’), meaning that this natural product contributes to hive defence. Due to its waxy nature and mechanical properties,
bees use propolis in the construction and repair of their hives - for sealing openings and cracks and smooth out the
internal walls [2, 3] - and as a protective barrier against external invaders or against weathering threats like wind and
rain. They also use bee glue to embalm the carcasses of dead intruders, thus avoiding their decomposition and
eliminating a potential source of microbial infections.
Propolis is a complex mixture composed of beeswax, resins and plant balsams, essential oils, pollen and some
organic and mineral compounds [1, 2]. It has been extensively employed by man since ancient times, especially in folk
medicine to treat or alleviate several maladies. Egyptians knew very well its anti-putrefactive properties and used bee
glue to embalm their cadavers. Incas employed propolis as an anti-pyretic agent. Greek and Roman physicians used it as
mouth disinfectant and as an antiseptic and healing product in wound treatment, prescribed for topical therapy of
cutaneous and mucosal wounds . These therapeutic applications were perpetuated in the Middle Age and among
Arab physicians. Listed as an official drug in the London pharmacopoeias of the 17
century, propolis became very
popular in Europe between the 17
centuries due to its antibacterial activity. In Italy, Stradivari used bee glue
as a violin varnish . In the end of 19
century, propolis was widely used due to its healing properties
 and in the
Second Global War it was employed in several Soviet clinics for tuberculosis treatment, due to the observed decline of
lung problems and appetite recovery. In the Balkan states it was one of the most frequently used remedies, applied to
treat wounds and burns, sore throat and stomach ulcer . The first scientific work with propolis, reporting its chemical
properties and composition, was published in 1908, and indexed to Chemical Abstracts (reference nº 192) . Later, in
1968, the first patent  was obtained to produce bath lotions in Romania. The following years saw an increase in
international patents, predominantly from the former USSR and satellite countries during the 1980s. In 2000 almost half
of the commercial licenses were Japanese , but in the last decades there were patents registered worldwide.
Nowadays, propolis is a natural remedy found in many health-food stores in various forms for ingestion or topical
use. It is still used in many regions of the world, including Japan and the European Union, either in cosmetics or as
popular alternative medicine for self-treatment of various diseases. Current applications of propolis include preparations
for cold syndrome (upper respiratory tract infections, common cold, flu-like infections), as well as dermatological
preparations useful in wound healing, treatment of burns, acne, herpes simplex and genitalis, and neurodermatitis.
Additionally, propolis is used in mouthwashes and toothpastes to prevent caries and treat gingivitis and stomatitis, and it
is widely used in cosmetics and in health foods and beverages. It is commercially available in the form of capsules
(either pure or combined with aloe gel, Rosa canina or pollen), extracts (hydroalcoholic or glycolic), mouthwash
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solutions (combined with lemon balm, sage, mallow and/or rosemary), throat lozenges, creams, powder and also in
more purified products from which the wax was removed.
Due to its medicinal and biological properties, propolis became the focus of great scientific interest during the last 30
years mainly envisaging its application in human and veterinary medicine, pharmacology and cosmetics.
2. Propolis characteristics, origins and chemical composition
Propolis is a lipophilic, hard and brittle material when cold but becomes soft, pliable, gummy and very sticky when
warm . It possesses a characteristic and pleasant aromatic smell and varies in colour from yellow-green, to red and
to dark brown depending on its source and age [3, 11].
As referred above, propolis is a complex mixture made from plant-derived and bee-released compounds. The
proportion of the various substances is variable and depends upon the place and time of collection  but, in general,
raw propolis is composed of around 50% resins, 30% waxes, 10% essential oils, 5% pollen and 5% of various organic
compounds [2, 12, 13]. More than 300 constituents were identified in different samples [11, 14], and new ones are still
being recognized during chemical characterization of new types of propolis [3, 15-19].
Many analytical methods have been used for separation and identification of propolis constituents and the substances
identified belong to the following groups of chemically similar compounds: flavonoids; benzoic acids and derivatives;
benzaldehyde derivatives; cinnamyl alcohol, and cinnamic acid and its derivatives; other acids and respective
derivatives; alcohols, ketones, phenols and heteroaromatic compounds; terpene and sesquiterpene alcohols and their
derivatives; sesquiterpene and triterpene hydrocarbons; aliphatic hydrocarbons; minerals; sterols and steroid
hydrocarbons; sugars and amino acids . As it may be expected, volatile compounds (produced by the source plants)
are present in low amounts . Sugars are thought to be introduced accidentally during the elaboration of propolis
and/or passage of bees over the resin. Some compounds are probably present in all propolis samples and contribute to
its characteristic properties. Others are represented in many samples of different origins, but a few others only occur in
propolis from particular plant species .
Many studies on the properties and composition of propolis have been made without knowing the plant(s) from
which the sample was obtained, or the sites where bees collected the material. However, the large number of studies
reporting to propolis chemistry allowed researchers to realize that its chemical composition is not only complex but also
highly variable, depending on the season and local flora at the site of collection as well as on the type of bees foraging
[3, 11, 17, 21]. The main visited plant species are poplars (Populus spp.), beech (Fagus sylvatica), horsechestnut
(Aesculus hippocastanum), birch (Betula alba), alder (Alnus glutinosa) and various conifer trees [1, 20]. The source
species can vary with geographical regions and determines propolis chemical composition . Indeed, there are
differences between propolis of temperate and tropical zones: while the materials used in temperate regions are
essentially bud exudates from different poplar trees , these are absent in tropical zones and bees use exudates of
other plants – mainly the leaf resin of Baccharis dracunculifolia - giving the resin a different composition. European
propolis contains the typical “poplar bud” phenolics: flavonoid aglycones (flavones and flavanones), phenolic acids and
their esters, whereas the major constituents of Brazilian propolis are terpenoids and prenylated derivatives of p-
coumaric acids [3, 22]. HPLC analysis of the phenolic compounds present in Populus nigra bud exudates clearly
support that this is the main origin for propolis in continental Europe, North America, West Asia, and New Zealand [1,
10, 23]. It is also reported that in areas where poplars are not native plants, such as in Australia and equatorial regions in
South America, bees gather exudates from Ambrosia deltoidea and Encelia farinose to make propolis. Phenolic
compounds of propolis from Venezuela have its origin in resin exudates of Clusia minor and Clusia major . The
most important biologically active constituents of propolis from different geographic locations and the corresponding
source species are represented in Table 1. Apart from plant exudates collected by bees, the compounds identified in
propolis are originated from other two sources: secreted substances from bee metabolism and also from materials
introduced during resin elaboration [1, 11]. In the absence of natural materials, bees may use some man-made products
like asphalt and mineral oils as substitutes [14, 24].
Poplar-type propolis is undoubtedly the most studied one but there are many other propolis types, as that found in
some Mediterranean regions (Sicily, the Adriatic coast) which has diterpenic acids as main components [25, 26]. More
recently, a new type of Brazilian propolis, popularly known as “red propolis”, was collected in northeast Brazil . Its
intense red colour and chemical composition make it different from the 12 types formerly classified by Park et al. .
In fact, Brazilian propolis is quite diverse in chemical composition, due to Brazil’s biological and climate diversity.
Brazilian red propolis, like Cuban red propolis, contains isoflavonoids which have been associated with a variety of
health benefits, including the relief of some symptoms in menopause, osteoporosis, hormonal cancer and prostate
cancer . In tropical countries of South America there are indigenous stingless bees which mix collected resinous
material with bee wax and soil, forming geopropolis [3, 14, 28].
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A. Méndez-Vilas (Ed.)
Table 1 Propolis types, geographic origin, main plant sources and chemical compounds [adapted from 11].
Biological activities are always present in propolis but they can be associated with completely different chemical
profiles in samples from different geographic and climatic zones . The principal compounds responsible for
propolis biological activities are flavonoids, aromatic acids, diterpenic acids and phenolic compounds, but very often
different propolis types have distinct main bioactive compounds (Table 1). Hegazi and co-workers  tested propolis
from Austria, Germany and France for antimicrobial activity and observed different activity spectra but some
similarities in qualitative composition. In propolis from Northern Argentina the highest antimicrobial and antioxidant
activity correlated with highest concentrations of flavonoids (pinocembrin) and phenolics . Many studies confirm
these results showing these compounds to be responsible for the antimicrobial and antioxidant properties of many
propolis types [22, 26]. Table 2 points up some of the compounds that have been correlated with specific bioactivities in
different propolis types , making evident that it is not possible to ascribe a certain property exclusively to one
individual component .
Table 2 Compounds responsible for four biological activities of different propolis types
Antitumor Antioxidant References
benzophenones Not tested Prenylated
and their esters
and their esters
Caffeic acid phenetyl
and their esters
Taiwanese Not tested Not tested Prenylated
The distinct chemical compositions of propolis from different origins led to the expectation that their biological
properties would be dissimilar, but this is amazingly untrue in many cases  and samples of different origins can
display identical biological activity. Kujumgiev and co-workers  observed that in spite of the phytochemical
differences found in propolis samples from different geographic locations, they all exhibited significant antibacterial
and antifungal activities, revealing that different combinations can result in a same bee glue´s biological activity. This
chemical redundancy, very common in nature, suggests that the antimicrobial activity is a vital property that bees must
guarantee independently from the geographic area they inhabit. Popova and collaborators [31, 32] used European,
Brazilian and Central American samples of propolis and showed that samples from Europe and Brazil had similar
activities despite the drastic differences in chemical composition, and that they were more active than Central American
propolis. It is clear that propolis research should include not only a chemical characterization but also combine different
biological tests . It also comes out from these observations that, probably, the differences in antimicrobial potency
and specificity of propolis sampled from different geographic regions might be related with the risk of emerging
(specific) microbial infections in those sites. Pushing forward this line of reasoning, it would be very interesting to
study in what extent propolis antimicrobial activity against specific pathogenic species might constitute an indicator of
the presence of those pathogens in the local or the risk of a certain disease.
type Geographic origin Plant source Main bioactive compounds
Europe, North America,
non-tropic regions of Asia
Populus spp., most
often P. nigra L.
Flavones, flavanones, phenolic
acids and their esters
propolis Russia Betula verrucosa Ehrh.
Flavones and flavonols (different
from poplar propolis)
B. dracunculifolia DC.
Prenylated p-coumaric acids,
propolis Cuba, Venezuela Clusia spp. Polyprenylated benzophenones
(Okinawa, Taiwan) Unknown C-prenylflavanones
propolis Canary Islands Unknown Furofuran lignans
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3. Propolis analysis
Propolis cannot be used directly as raw material and a simple fractionation to obtain compounds is difficult due its
complex composition. The usual procedure is the use of a solvent , which should remove the inert material and
preserve the desired compound(s). As the composition of propolis primarily depends upon the vegetation from where it
was collected but secondarily upon the methods used for extraction , the solvent should be carefully chosen .
The principal solvents used for extraction of bioactive compounds, and the correspondent classes of chemical
compounds extracted are depicted in Table 3.
Table 3 Solvents used for active component extraction. Compounds in bold are commonly obtained only in one solvent
[adapted from 34].
A routine and common procedure consists to extract the fraction soluble in alcohol, called “propolis balsam”,
discarding the insoluble fraction or wax fraction . Ethanol is the most common solvent choice, but other solvents
have also been used for separation and identification of many constituents in propolis [35-37]. Studies regarding the
evaluation of propolis´ bioactivities have been performed using mainly ethanol extracts of propolis (EEP) or water
In the last decade the idea that propolis has a complex but more or less constant chemistry, has radically changed. A
new paradigm arose, supported by the analysis of numerous propolis samples collected in different seasons and from
different geographic regions, leading to the discovery that the chemical composition of bee glue is highly variable. This
diversity in propolis chemistry is reflected on its pharmacological properties and demands for standardization . In
order to be accepted officially into the main stream of the healthcare system, chemical standardization is effectively an
obligatory criterion. Such standardization could be achieved by formulating different propolis types according to plant
source/chemical profile but for that it is essential to have detailed and reliable comparative information on every type of
biological activity and chemical data. Propolis is a widely accepted product and has an established safety profile.
However, it is also a known contact allergen and may seldom induce some adverse reactions as revealed by some
reported cases of allergy and contact dermatitis [38-40]. Furthermore, when natural plant materials are absent for
propolis elaboration, bees may use some man-made products like asphalt and mineral oils as substitutes . This can
introduce some unsafe substances such as lead (Pb) and other metals like copper (Cu), cadmium (Cd) and zinc (Zn)
which can contribute to the increase of propolis toxicity . International markets are also very demanding in terms of
trace compounds, heavy metals and environmental pollutants , and the development of standardized manufacturing
procedures is needed. Propolis users, in particular the companies that produce propolis formulations, need to know the
characteristic concentrations of its constituents to guarantee a good product quality and a reasonable degree of
bioactivity, and current protocols do not take into account the variable composition and pharmacological properties of
propolis . Additional tests are needed to fully investigate the biological effects of propolis, not only for those
bioactive compounds already described but also for others, considering the chemical diversity of this natural product.
Comparative studies on propolis collected from a wide range of countries are thus crucial for linking its provenance to
certain bioactivities and hence ensuring that the beneficial properties of propolis are used on a rational base and more
effectively by the public.
The analysis of all the biologically important individual components of propolis is often a tedious, time consuming
and expensive procedure. It can be avoided in cases when the plant origin and the qualitative composition of the
propolis are known. In such case, the rapid and low-cost determination of the quantitative chemical profile of a sample
by measuring the concentration of groups of compounds similar in chemical nature (e.g. total flavonoids) is convenient
and reasonable. Popova et al.
 developed and validated rapid, low-cost spectrophotometric procedures, which
demonstrated that measuring the concentrations of groups of active compounds instead of individual ones could be an
adequate approach in the case of propolis. Later on, the same researchers argued that measurement of minimal
inhibitory concentration (MIC) should be an obligatory element in propolis quality control, due to the complex
synergistic effects of different propolis constituents.
Research on polyphenols (flavonoids and related phenolic acids) has been prompted by their noticeable beneficial
effects on health. Flavonoids aroused great interest after they had been found to have effects in inhibiting the copper-
catalyzed oxidation of low-density lipoprotein, inhibiting platelet clotting and arachidonate metabolism, reducing liver
Water Methanol Ethanol Chloroform Dichloromethane Ether Acetone
A. Méndez-Vilas (Ed.)
injury from peroxidized oil, and having cancer-chemopreventive properties [42, 43]. Several methods have been
developed to analyse polyphenols: thin-layer chromatography (TLC), gas chromatography (GC), high-performance
liquid chromatography (HPLC), HPLC-mass spectrometry (HPLC-MS), and capillary electrophoresis (CE).
Furthermore, liquid chromatography (LC)-MS technique is able to separate each single component in complex mixtures
and to perform their identification and quantification . Due to these several advantages, LC-MS has gained
widespread interest and became a reference technique for the determination of pharmacologically interesting
compounds in biological matrices. Accurate assessment of the contents of bioactive compounds in extract samples
requires the validation of certain analytical parameters such as precision, recovery, linearity and detection limits.
Therefore, on-line HPLC-electrospray ionization (ESI)/MS analysis, usually used for commercial pharmaceutical
preparations, constitutes an alternative to obtain propolis fingerprints and a reliable identification of a large number of
propolis polyphenolic components .
4. Biological activities
Independently from the plant source (plant species and geographic origin) and chemical composition, a biological
activity of bee glue has always been reported [26, 45], in particular the antimicrobial activity. This is probably the
reason why bee glue plays such an important role in the hive: it is a “chemical weapon” against pathogenic
microorganisms, a constant threat to the vital sanitary status of this crowded and busy “city”, vulnerable to the invasion
of an array of enemies and to the proliferation of diseases. In spite of this universal function and due to plant diversity,
there are different propolis types, which contain numerous chemical constituents responsible not only for the
antimicrobial property but also for other valuable bioactivities . These biological activities include: antibacterial
[17, 26, 46, 47], antifungal [26, 48, 49], antiviral [50, 51], antiprotozoan [52-54], antitumour [39, 40, 55, 56], anti-
inflammatory [57-59], local-anesthetic , antioxidant [61-63], immunostimulating [64-65], cytostatic  and
hepatoprotective [66, 67].
The active factor(s) in propolis that are responsible for its many biological properties frequently remains to be fully
defined and varies with propolis sample, dosage, and the extraction solvents used . Flavonoid and esters of phenolic
acids are generally regarded as bioactive compounds
but, as a natural mixture of organic compounds, it may well be that
the ratio of the combined reagents in propolis is important for its effect.
One of the first biological activities to be recognized and probably one of the most important properties of propolis is
the antimicrobial activity, especially against bacteria. Several studies have been performed to evaluate this property
against a large panel of Gram-positive and Gram-negative bacteria (Table 4), either aerobic or anaerobic, from
laboratory collections or isolated from clinical samples, using propolis of different origins and chemical composition
and exploring different experimental approaches.
The disc diffusion method is one of the most popular methods used to evaluate this activity. A suspension of a
sensitive indicator microorganism is inoculated on agar plates by spreading homogeneously on its surface, and blank
paper discs containing the sample to be tested for antimicrobial activity are placed on top. After an adequate incubation
period at optimal temperature, antibacterial activity is determined by measuring the diameter of the growth inhibition
zones (inhibition halos) in the agar layer surrounding the disc . Some authors argue that this laborious method is
unreliable for comparing bioactivities, as results are influenced by the solubility and hence the diffusivity of the
individual constituents in agar, proposing the use of another methodology also commonly used for the same purpose:
the dilution method. In this procedure, propolis samples are serially two-fold diluted and a fix volume is added to liquid
or solid medium, making a concentration series. A bacterial inoculum is subsequently added to each experimental
condition and the occurrence of growth is analysed after incubation at optimal conditions. Broth microdilution is
considered a good method for a rapid and simultaneous screening of multiple samples; it is suitable for comparing
propolis extracts and gives more consistent results. Additionally, it allows the determination of the minimal inhibitory
concentration (MIC) and the minimal bactericidal concentration (MBC) which are, respectively, the lowest
concentration that inhibits visible bacterial growth and the lowest concentration that kills bacteria [46, 80]. Ideally, a
broad range of operational concentrations should be prepared in order to obtain empirical data suitable for dose-
response mathematical modelation. In this way more information could be drawn from the experiment, being possible
to estimate other important parameters such as the inhibitory concentration IC
, which corresponds to the
concentration that induces a 50% reduction in growth. Less common in propolis research but also used to evaluate
antimicrobial activity of several compounds is bioautography. Briefly, thin layer chromatography plates where propolis
samples were eluted are covered with agar suspensions of the microorganism which sensitivity is going to be tested.
Antibacterial activity is visualized as clear areas after proper incubation.
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Table 4 Bacteria screened for sensitivity to propolis in antimicrobial tests.
Gram-positive References Gram-negative References
[16-18, 26, 28,
47, 69, 70, 77]
[16-19, 26, 28, 31,
47, 69, 70, 73, 77]
(S. auricularis, S. capitis, S.
epidermidis, S. haemolyticus, S.
hominis, S. mutans, S. warnerii)
[19, 28, 69, 70, 73]
(S. enteritidis, S. typhi, S. typhimurium)
[16, 47, 71, 77-
(S. cricetus, S. faecalis, S.
pneumioniae S. pyogenes, S.
haemolyticus, S. mutans, S.
sobrinus, S. viridians)
[16, 19, 30, 69, 71,
[17, 28, 46, 47,
Data from numerous studies concerning antibacterial properties of propolis support the fact that propolis is active
mainly against Gram-positive bacteria and either displays much lower activity against the Gram-negative ones or is
inactive at all [11, 17, 26, 45-47, 49, 58, 77, 80]. Such results can be seen in the work of Kujumgiev et al.  who
tested propolis samples from different geographic regions (tropical and temperate zones) against Staphylococcus aureus
and Escherichia coli. All the extracts displayed significant antibacterial activity against S. aureus but none was active
against E.coli. Although it can be argued that only two species were tested, it is also relevant that all the 12 samples
tested, from so different origins, showed the same effect. Grange and Davey  reported that ethanol extracts from
propolis (EEP) completely inhibited the growth of S. aureus, Enterococcus spp. and Bacillus cereus, partially inhibited
Pseudomonas aeruginosa and E. coli growth and had no effect on Klebsiella pneumoniae. Sforcin et al. 
investigated the antibacterial activity of Brazilian propolis collected during the four seasons and observed that low
concentrations inhibited the growth of Gram-positive bacteria and that higher concentrations of EEP were needed to
inhibit Gram-negative bacteria. Interestingly, no significant differences were registered on the survival curves of S.
aureus and E. coli after incubation with propolis collected at different times. It could be expected that propolis produced
in different seasons, and therefore from potentially different plant species or plant organs, exhibit significant chemical
variation but, as climatic diversity is also an important factor, it may be the case that the sampling area might not reveal
a significant climatic variation during the year and so it may have a more or less constant flora.
A variety of studies have been performed with bacteria of odontological relevance, which is one of the most
prominent areas of propolis application. Dental plaque is related with colonization of oral microorganisms and the
accumulation of extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus
spp. . Park et al.  tested Brazilian propolis samples collected in various regions and noticed that all the samples
inhibited the enzyme activity. Also, several periodontal disease-causing anaerobic bacteria are susceptible to propolis
aqueous-ethanolic extract, again the Gram-negative being the most resistant to propolis action [44, 75, 76]. Some
authors suggested that the increased resistance of Gram-negative bacteria could be due to the presence of plasma
membrane efflux pumps that would prevent intracellular entry of propolis constituents, or promote their extrusion from
the cell, or even because propolis contains many plant-derived resin constituents which are secreted to protect plants
from Gram-positive pathogens mostly . Varroa mites are parasites that can destroy the hive and which harbours
predominantly Gram-positive bacteria , a fact that could be another ecological rationale for this “antiseptic” activity
against Gram-positive microbes.
Propolis antibacterial activity has been mainly correlated with flavonoids, with galangin, pinocembrin and
pinostrobin being recognized as the most effectives . However, there are reports of propolis samples containing
only traces of flavonoids but displaying an antibacterial action . Ferulic and caffeic acid, prenylated coumaric acid
and benzophenone derivatives or diterpenic acids are also bioactive compounds [1, 2, 26, 28, 32, 83] but the exact
A. Méndez-Vilas (Ed.)
mechanism of antimicrobial action still remains to be elucidated and has been subject of only a few publications.
Through electron microscopy and micro-calorimetric assay, it was shown that EEP interferes with the division of
Streptococcus agalactie through the formation of pseudo-multicellular forms, cytoplasm disorganization, protein
synthesis inhibition and cell lysis . Many bacteria are effectively killed by flavonoids. However, the primary targets
of flavonoids, the eicosanoids, do not appear to be formed by bacteria as the involved enzymes are only present in
eukaryotic cells. Prokaryotic do however contain metalloenzymes, such as phosphatases, and the heavy metal atoms
form a strong ligand complex with flavonoids. The bactericidal effect of flavonoids may therefore be originated from a
metabolic perturbation in ion channels as a result of impairment in phosphorylation/dephosphorylation reactions [70,
83]. Caffeic acid, benzoic acid, and cinnamic acid probably act on the microbial membrane or cell wall sites as well,
causing functional and structural damages [11, 83]. Some diterpenes and phenolic compounds possess activity against
Helicobacter pylori  a Gram-negative bacteria that causes peptic ulcer disease and gastric cancer.
Until now, no single propolis component has shown to possess antibacterial activity higher than that of the total
extract . Some authors attribute the highly complex and variable composition of propolis as a reason for its antimi-
crobial activity and the data gathered so far suggests that it can be linked to multiple targets, with several constituents
acting in synergy [2, 51, 73, 83, 84]. Propolis affects cytoplasmic membrane, inhibits bacterial motility and enzyme
activity, exhibits bacteriostatic activity against different bacterial genera and can be bactericidal in high concentrations
. The antibacterial efficacy of EEP increases at higher temperature (37 ºC) and at acidic pH (pH 5.0) . Propolis
has a multiple action against many virulence factors of Gram-positive bacteria of clinical interest: staphylococcus’s
virulence factor coagulase is completely suppressed, lipase strongly reduced and a dose-dependent prevention of
biofilm formation is evident in the presence of EEP . Although in vivo observations are still missing, this reduction
of microbial virulence factors is undoubtedly an interesting target in the treatment of Gram-positive infections.
Synergism between EEP and antibiotics with a clear reduction of MIC values for several strains has also been reported
[73, 78, 80).
The evaluation of antifungal activities of propolis extracts is normally performed using the methods already described
for bacteria. The disk diffusion assay has been used to assess the sensitivity of several yeasts and fungi to ethanol
extracts of different propolis samples. Although antibacterial activity is more relevant than the antifungal properties of
propolis, many studies have reported the susceptibility of clinically important yeasts belonging to Candida genera [26,
49, 80] such as Candida albicans [16, 18], as well as the sensitivity of some filamentous fungi, mainly dermatophites
. Longhini and co-workers  showed that propolis has antifungal activity in dermatophytes even in small
concentrations, present low toxicity and so that it can be used topically. Various yeast species isolated from
onychomycosis, including Saccharomyces cerevisiae and Trichosporon sp. are susceptible to propolis . European
propolis samples have a fungicidal effect against Candida, Microsporum, Mycobacteria, Trichophyton, Fusarium and
other dermatophytes . An antifungal activity of propolis was also observed in some plant fungi in vitro. In the case
of Brazilian EEP, which was tested against Candida sp., it was shown that C. tropicalis was the most susceptible
followed by C. albicans, C. guilliermondii and C. parapsilosis .
The fungicidal effect was associated with the presence of flavonoids  and other phenolic components such as for
antibacteria properties. Differences in antifungal activity of propolis extracts can again be attributed to the differences in
chemical composition and concentration of propolis compounds [17, 89]. As for antibiotics, a synergistic effect with
conventional antimycotic drugs was observed .
Another important biological property already ascribed to propolis is the antiprotozoan activity. This property is
evaluated by an in vitro growth inhibitory effect on a culture of parasites after incubation in the presence of different
concentrations of propolis. Population density is estimated upon centrifugation and the growth inhibitory effect is
determined comparing the values obtained for cultures treated and untreated with propolis.
Several publications reported the effect of European propolis on protozoa that cause diseases in humans and animals
such as trichomoniasis, toxoplasmosis, giardiasis, Chagas disease, leishmaniasis and malaria. Indeed, antiprotozoan
activity has also been reported on Giardia lamblia, Trichomonas vaginalis, Toxoplasma gondii, Leishmania donovani
and Trypanosoma cruzi [90, 91]. Propolis preparations were classified as a good coccidiostat against Chilomonas
paramecium . An antiprotozoan activity of EEP was reported against G. duodenalis , as well as an inhibitory
effect in trophozoite adherence and a reduction of flagella beating frequency in great part of trophozoites.
Antiprotozoan activity was verified in experimental Trypanosoma cruzi-infected mice treated with 50 mg/kg body
weight/day of Bulgarian EEP, leading to a decrease in parasitemia , and in experimental animals infected with
Eimeria magna, E. media and E. perforans . As T. cruzi is the etiologic agent of Chagas disease, an endemic
parasitosis that infects 16–18 million people in Latin America , these results are quite enthusiastic. Propolis also
seems to have a prophylactic effect against malaria in endemic areas in Brazil .
A. Méndez-Vilas (Ed.)
The antiviral activity is normally assessed with the cytopathogenic effect (CPE) reduction assay. Confluent cell
monolayers are prepared in plastic plates, infected with virus and incubated for the required period of time at an
appropriate temperature, after which cells are microscopically observed for a cytopathogenic effect.
There are few data available concerning antiviral effects of propolis but the studies performed have shown that
propolis from various geographic regions displays significant antiviral activity, acting at different levels and interfering
with the replication of some viruses . The in vitro effect of EEP was evaluated on several DNA and RNA viruses
including herpes simplex type 1, herpes simplex type 2, adenovirus type 2, vesicular stomatitis virus and poliovirus type
2 [50, 51]. The results provided evidence that propolis is very active in vitro against poliovirus and herpes viruses,
whereas vesicular stomatitis virus and adenovirus are less susceptible. The inhibition of poliovirus propagation was
clearly observed. Besides this effect on virus multiplication, a virucidal action on the enveloped viruses herpes simplex
and vesicular stomatitis virus was also detected. Virus inactivation was time and dose-dependent. It was reported that
propolis affected the replication of influenza viruses A and B, vaccinia virus and Newcastle disease virus. Harish and
co-workers  found that propolis suppressed HIV-1 (human immunodeficiency virus) replication. Antiviral activities
of propolis samples from Egypt reduced the infectivity mean titers of the Bursal Disease virus and Reo-virus , and
propolis samples from different geographic origins showed antiviral activity against Avian influenza virus .
Flavonoids and aromatic acids derivatives are responsible for the antiviral activity of propolis extracts . Some
flavonoids (baicalin) have inhibitory effect on HIV infection and replication as showed by in vitro studies. In the
inhibition of Amazon parrot herpes virus, König and Dustmann  verified that luteolin was more active than
quercetin, but remarkably less than caffeic acid. Some esters of substituted cinnamic acids found in propolis also proved
to have antiviral properties: isopentyl ferulate significantly inhibited the infectious activity of influenza virus A and 3-
methylbut-2-enyl caffeate showed strong inhibition on herpes simplex virus type 1 growth [50, 51]. Vaccinia and
adenovirus were more sensitive than polio and parainfluenza virus to the antiviral effect of caffeic acid, which displays
a weak activity against influenza virus.
This broad antimicrobial activity of propolis targeting a wide spectrum of phylogenetic different taxa and life forms,
from virus thru prokaryotes to single cell eukaryotes and to more complex multicellular organisms, like the protozoa
and filamentous fungi, is consistent with an important non-specific function of ecological relevance: the protection of
hive community from potential microbial infections, that could rapidly propagate in such a densely colonized and
confined space, with potential devastating results.
4.5 Antioxidant activity and associated biological properties
Free radicals are highly reactive species that can damage cellular components, such as proteins, nucleic acids and lipids,
and are implicated in a variety of diseases. Their reactivity is usually neutralized in the body by antioxidant enzymes
and nutrient-derived antioxidant molecules, which protect humans from deleterious oxidative processes. Propolis is
notable for its antioxidant properties, only surpassed by those of green tea, and is more active than the rest of the
beehive products in what concerns this property . The antioxidants present in propolis [98-101] play a great role in
its immunomodulatory properties . It was reported that propolis increases the cellular immune response through
the increase of mRNA for interferon-γ and activates the production of cytokines .
One of the most common used techniques to evaluate antioxidant potential is based on the depletion of free radicals
by the addition of scavenger compounds. Measurements of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical consumption
are related to the intrinsic ability of a substance or a complex mixture to donate hydrogen atoms or electrons to this
reactive species in a homogeneous system.
The relatively strong antioxidant effects exhibited by EEP from different geographic origins (Argentina, Australia,
China, Hungary and New Zealand) were correlated with high polyphenol and flavonoid contents, particularly
kaempferol and phenethyl caffeate . Similar results were obtained with Turkish propolis and with samples from
various regions of Korea . DPPH free radical-scavenging activity of Korean EEP was higher than that of butylated
hydroxytoluene (BHT), used as control. In general, synthetic antioxidants showed better antioxidant properties than
EEP, but at higher concentrations the reducing power of EEP and of artificial antioxidants were similar. Ferulic acid,
quercetin, caffeic acid, prenylated compounds, apigenin and also galangin, p-coumaric and CAPE were identified as
bioactive compounds responsible for antioxidant potential in different propolis samples [44, 98-101]. Oyaizu et al. 
reported that α-tocopherol is contained in almost all propolis samples and correlates with its antioxidative effect.
4.6 Antitumor and cytotoxic activity
Antitumor activity, including cytotoxicity, was reported for EEP [39, 40, 55, 56]. Different methods allow
determination of cytotoxic effects in vitro but, normally, cells are maintained in appropriate medium and then cultured
in the presence of different concentrations of propolis extracts. Cellular viability is then assessed using the MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide] or Trypan Blue Exclusion assays.
Some new compounds responsible for these properties such as diterpenic acids were isolated from propolis.
Clerodane-type diterpenes PMS-1 exhibited cytotoxicity towards human lung carcinoma HLC-2 and human cervical
A. Méndez-Vilas (Ed.)
carcinoma HeLa cells . Coniferyl aldehyde, betuletol, kaempferide and ermanin isolated from Brazilian propolis
showed potent cytotoxicity towards human HT-1080 fibrosarcoma and murine colon 26-L5 carcinoma cells, having
effective dose (ED
) values of 10 µg/ml . The new prenylflavanones propolin A and propolin B from Taiwanese
propolis exhibit cytotoxic properties towards human melanoma, C6 glioma, and HL-60 cell lines, inducing apoptosis
with DNA fragmentation . Propolin H isolated from Taiwanese propolis samples inhibited the proliferation of
human lung carcinoma cell lines through induction of G1 phase cell cycle arrest . CAPE has been identified as one
of the major active compounds in propolis with chemopreventive and antitumor properties
 without being cytotoxic
to normal cells . A direct relationship between the cytotoxic effects of CAPE and the induction of DNA
fragmentation and apoptosis was established by Su et al. . In what concerns flavonoids, the assessment of its
activity against HeLa cells revealed the following susceptibility order: quercetin had the strongest antitumor activity,
followed by rhamentin and galangin .
Natural resistance to tumour development has been associated with the cytotoxic activity of natural killer (NK) cells
, a lymphocyte subpopulation that shows lytic activity principally towards several types of tumour and virus-
infected cells. Sforcin et al.  found an increase of NK activity in spleen cells of propolis-treated animals.
4.7 Other bioactivities
Many other biological and pharmacological properties of propolis have been described in several studies , including
tissue regenerative properties, anti-inflammatory effects, immunogenic properties, liver detoxifying action,
hepatoprotective activity, choleretic and antiulcer action in vitro. The hepatoprotective effect appears to be due to the
presence of dicaffeoylquinic acid derivates and flavonoids . Cardioprotective , neuroprotective  and
radioprotective  effects were also reported. Propolis seems to lower cholesterol levels and blood pressure making
possible its use in the prevention and treatment of atherosclerosis . Furthermore, Maruyama and collaborators 
suggest that EEP of Brazilian green propolis and its main constituents may be useful for prevention of hypertension.
Propolis has anaesthetic activity with effects similar to those of cocaine . It also kills the ectoparasitic mites Varroa
destructor , which attack honeybees causing the varroatosis disease.
5. Propolis applications and future challenges
One of the most ancient applications of propolis is in odontology to reduce the incidence of dental caries [30, 74] and in
dermatology due to its capacity of wound healing and promotion of tissue regeneration . Propolis is also claimed to
have beneficial effects on otorhinolaryngologie and in gynecological diseases  as well as in stomatology  and
geriatry. The various uses of propolis in clinical trials show that its therapeutic efficacy lies mainly in diseases caused
by microbial contaminations.
The broad spectrum of propolis biological properties, the long history of its use and safety profile, together with the
results that are being observed in preclinical studies, provide a rationale for several applications in human and
veterinary medicine as well as in pharmacology, which must be studied in clinical settings. Propolis synergism with
antibiotics could allow the reduction in the dose of selected antimicrobials, potentiating the antimicrobial therapy. This
is particularly important in the light of the widespread emergence of antibiotic resistant strains and of opportunistic
pathogens, which demand for novel therapeutic agents and strategies, but the exact propolis compounds that are
involved in the mechanisms of synergistic interaction with other drugs must be identified.
The increasing interest towards natural therapies, effective and healthy pharmacological compounds is obviously a
stimulus for propolis research. However, new challenges are emerging in this field. As already referred, propolis
chemical composition and pharmacological activity may vary widely from region to region , and potential medical
applications of propolis have led to the need of standardization, and of origin and quality control. Moreover,
bioactive(s) compound(s) remain to be identified or fully defined in many propolis types and studied samples.
Subsequent studies will be needed in order to isolate and identify the main active compound(s) through bio-guided
fractionation, to confirm if individual isolated compound(s) can reproduce propolis effects and, finally, to disclose the
molecular targets and mechanisms underlying the biological activities.
Propolis research continues to surprise scientists and remains a fascinating subject for further studies and
applications. The continuous discovery of new compounds demonstrates that the search for new promising bioactive
compounds and biological activities is still a hot topic. But propolis research should not be restricted to this applied
perspective of natural products; the comprehensive understanding of this bee-made product at the ecological level, its
roles in hive integrity and sanitary conditions, and eventually in bees health and disease control, is no less stimulating.
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