Arch Dermatol Res (2002) 293 : 594–596
DOI 10.1007/s00403-001-0275-x
S H O RT C O M M U N I C AT I O N
N. Ledón · A. Casacó · R. González · J. Bracho ·
A. Rosado
Assessment of potential dermal and ocular toxicity and
allergic properties of an extract of red propolis
Received: 26 April 2001 / Revised: 11 July 2001 / Accepted: 17 October 2001 / Published online: 11 December 2001
© Springer-Verlag 2001
Keywords Propolis · Allergy · Dermal toxicity · Ocular
toxicity
Introduction
Propolis is a resinous mixture collected by bees from the
buds of trees which they use to seal their hives (Simonetta
et al. 1997). The use of propolis in folk medicine has in-
creased substantially in the last two decades and it is rec-
ommended for the treatment of various diseases. Propolis
exerts antimicrobial, antiinflammatory, analgesic, antiox-
idative and antipsoriatic effects (González et al. 1994;
Ledón et al. 1996; Pascual et al. 1994). However, several
European authors (Burdock 1998; Callejo et al. 2001;
Hashimoto et al. 1988; Hausen et al. 1987; Young 1987)
have reported significant side effects, particularly allergic
reactions. To date more than 500 compounds have been
identified in European propolis (Hegyi et al. 1990) and
their allergic properties have been ascribed to the content
of caffeic acid esters and flavonoid aglycones (Hausen
and Wollenweber 1988; Hausen et al. 1992). However,
propolis obtained in tropical countries does not contain
these compounds (Bracho et al. 1996; Cuesta et al. 1999;
Greenaway et al. 1991).
Taking into account the previous findings, we decided
to determine the sensitizing properties of an extract of
Cuban red propolis which is mainly obtained from the
coast plant Rhizophora mangle. We also investigated the
ocular and dermal toxicity of this extract.
N. Ledón (✉) · R. González · A. Rosado
Centro Nacional de Investigaciones Científicas,
PO Box 6412, Havana, Cuba
e-mail: nuris@sis.copextel.com.cu
A. Casacó
Centro de Inmunologia Molecular, Havana, Cuba
J. Bracho
Estación Experimental Apícola. Havana, Cuba
Materials and methods
Animals
All animals were purchased from the National Center for Labora-
tory Animal Production (Cenpalab, Havana, Cuba). They were
housed in an environmentally controlled room under constant air
humidity with a 12-h light-dark cycle. They were fed standard lab-
oratory diet and had access to drinking water at libitum. The pro-
cedures were approved by the ethical committee for animal exper-
imentation of National Center for Scientific Research (CNIC) and
the principles of laboratory animal care were followed.
Preparation of propolis extract
The Cuban propolis used in this study was classified from its sep-
aration pattern by thin-layer chromatography and was named
Propolis R or red. It was collected from beehives in Consolación
del Sur, Pinar del Rio Province. The propolis extract was obtained
according to the method described by Alvarez et al. (1989). Volatile
constituents from this propolis were isolated and analyzed by gas
chromatography-mass spectrometry under the conditions recom-
mended by Bracho et al. (1996).
Dermal toxicity test in rabbits and guinea pigs
Two groups of six New Zealand rabbits (2–2.5 kg) and Hartley
guinea pigs (325–400 g) were shaved on the dorsal area. The right
skin area was left intact while the other side was abraded superfi-
cially without damaging the dermis in a random pattern. Propolis
extract (0.5 g in 0.5 ml 70% ethanol) or the ethanol vehicle alone
was applied to each site which was then covered with a 2-cm four-
layered gauze pad. The entire trunk of the animal was wrapped
with 6-cm wide adhesive tape to fix the patches and this was held
in place with tape. Care was taken not to restrict the respiratory
and abdominal movements of the animal. The patches were re-
moved after 24 h and the results were evaluated according to the
method described by Draize et al. (1944).
Ocular toxicity in rabbits
The eyes of nine New Zealand rabbits (1–1.5 kg) were examined
with 2% fluorescein 24 h prior to being tested. Red propolis (0.1 g)
was instilled into the conjunctival sac of the right eye and the lids
were held together for 1 s. The other eye was the control. In six of
the rabbits the eyes were left unflushed after instillation, and in the
remaining three rabbits the eyes were flushed 30 s after instillation
 595
for 1 min. Readings were made of the challenge site 24, 48 and 72 h Table 2 Allergic reactions (erythema) to Cuban red propolis in
after instillation. The intensity of erythema and edema were scored guinea pigs. All doses of propolis produced significant allergic
using the scales of Draize et al. (1944). reactions (P<0.05) compared with treatment with vehicle (70%
ethanol), which produced no reactions. Values are number of ani-
mals showing reaction/total number of animals
Allergy test in guinea pigs
Propolis dose (%) Gradea Day 1 Day 2 Day 3
Induction of sensitization
15 1 3/7 5/7 7/7
The nuchal area of Hartley guinea pigs (325–400 g) was shaved, 2 3/7 2/7
depilated and stripped. Ethanolic solutions of propolis (5, 10 or 3 1/7
15%) or the ethanol vehicle alone were applied 15 min after strip-
ping using occlusive patches. Five treatments were given during a 10 1 4/7 6/7 7/7
10-day period. 2 3/7 1/7
5 1 4/7 7/7
2 3/7
Elicitation of contact sensitivity
aGrade 1, slight reaction; grade 2, moderate reaction; grade 3, se-
After 21 days from the last sensitization a 2.5×2.5-cm area of the vere reaction
lumbar region that had not been previously exposed to the sub-
stances under test was shaved and depilated, and 0.1 ml of test sub-
stance was applied. (The challenge doses were the same as used
for sensitization.) The reactions were read at 24, 48 and 72 h. first day, while the medium and lowest doses produced
only slight reactions (Table 2).
Statistics

In all experiments examinations were conducted using a blind pro- Discussion

cedure by which the researcher did not know what kind of treat-
ment the evaluated animals had received. Significances of differ- The capacity of propolis to induce allergenic effects has
ences between two groups were evaluated by Student’s t-test. previously been reported (Hausen et al. 1987; Marcucci
When more than two groups were involved, the homogeneity of
the variance was evaluated by Bartlett’s method. Duncan’s multi- 1995; Young 1987). However, the propolis extracts used
ple range test was performed if the variance was homogeneous, in these studies were collected in other geographical areas
and Scheffe’s test if there was no homogeneity. with different climate and flora. These allergenic effects
of propolis have been ascribed to esters of aromatic acids,
e.g. dimethylallyl caffeic acid ester (Hashimoto et al. 1988;
Results Hausen et al. 1987), and some flavonoids, e.g. tectochrysin,
although the latter is considered a weak sensitizer (Mar-
None of the guinea pigs treated with propolis showed ery- cucci 1995). However, research in our department on the
thema or edema at 24 h in the dermal toxicity test (results chemical composition of tropical red propolis collected in
not shown). The propolis extract was not irritant in the Cuba has not shown the presence of dimethylallyl caffeic
dermal toxicity test (primary cutaneous irritation 0.58) or acid ester or flavonoids in the samples tested, indicating
ocular toxicity test (score 7) in rabbits (Table 1). In the that other compounds, such as the naphthoquinones ane-
contact allergy test, propolis extract produced a positive tol, eugenol and methylisoeugenol, and elemicine identi-
but moderate response that was dose-dependent. All doses fied by gas chromatography-mass spectrometry might be
produced erythema, but edema or other reactions were not the cause of the allergic effects (Hausen et al. 1992).
seen. The highest dose produced a mild erythema on the Research using different fractions of red propolis is in
progress in our laboratory with the aim of determining
which substance or substances are able to induce the al-
Table 1 Primary cutaneous irritation and ocular toxicity of Cuban lergic effects in experimental animals. Propolis has been
red propolis in rabbits. Cutaneous reactions were all erythema; no used in folk medicine in Cuba for many years for the
edema was seen. Values are number of animals showing reac-
tion/total number of animals
treatment of various diseases and its use has recently in-
creased substantially. However, a very low occurrence of
Primary cutaneous irritation Ocular toxicity allergic reactions induced by propolis in humans has been
reported in Cuba. Therefore, additional preclinical and
Undamaged Damaged Unflushed Flushed
skin skin eye eye epidemiological studies are necessary to elucidate these
aspects. Meanwhile, caution must be exercised in the use
Day 1 Day 3 Day 1 Day 3 Day 1 Day 2 Day 1 Day 2 of propolis formulations in allergic and asthmatic patients.
Propolis 0/6 0/6 6/6a* 6/6b* 1/6c 0/6 1/3c 0/3
Vehicle 0/6 0/6 0/6 0/6
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