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Acute and sub-acute oral toxicity of Brazilian

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 Journal of Ethnopharmacology 170 (2015) 66–71

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Acute and sub-acute oral toxicity of Brazilian red propolis in rats

Rafaela Oliveira da Silva a, Valléria Matos Andrade a, Ester Seixas Bullé Rêgo a,b,c,
Grace Anne Azevedo Dória a,b,c, Bruno dos Santos Lima b, Francilene Amaral da Silva b,
Adriano Antunes de Souza Araújo b, Ricardo Luiz Cavalcanti de Albuquerque Júnior a,b,c,
Juliana Cordeiro Cardoso a,c, Margarete Zanardo Gomes a,c,n
a
Tiradentes University, 300 Murilo Dantas Ave, Farolândia 49032-490 Aracaju, SE, Brazil
b
Department of Pharmacy, Federal University of Sergipe, Marechal Rondon Ave, Cidade Universitária, CEP 49100-000 São Cristóvão, SE, Brazil
c
Research and Technology Institute (ITP), 300 Murilo Dantas Ave, Farolândia 49032-490 Aracaju, SE, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Propolis is a bee product widely used in folk medicine due to its
Received 30 December 2014 numerous pharmacological properties. However, samples from different regions can differ in chemical
Received in revised form composition, effectiveness, and side effects. Despite the widespread use of Brazilian red propolis, which
4 May 2015
is an isoflavone-rich variety, its toxicity has not been carefully studied.
Accepted 4 May 2015
Available online 13 May 2015
Aims of the study: To assess the acute and sub-acute toxicity of the hydroethanolic extract of red propolis
(HERP) administered orally to rats.
Keywords: Materials and methods: HERP for the acute (300 mg/kg) and sub-acute (10, 100 and 200 mg/kg) toxicity
Red propolis studies was administered orally to rats according to OECD Guidelines 420 and 407, respectively. Clinical
Acute toxicity
signs were identified, and hematological and biochemical analyses were performed. Water and food
Sub-acute toxicity
uptake as well as body and organ weights of animals were recorded.
Isoflavone
Hematological parameters Results and conclusions: The acute study revealed no lethal effects at 300 mg/kg of HERP, but toxic signs
Biochemical parameters were observed, as HERP had an LD50 of more than 300 mg/kg, indicating a warning. The most toxic
signals in sub-acute studies were observed in males at a dose of 200 mg/kg HERP. These results suggest
estrogen-like activity, possibly from the isoflavones in HERP.
& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The recorded use of propolis in folk medicine dates back to about
300 BC. Its widespread use is related to numerous properties, such as
antibacterial, antiseptic, anti-inflammatory, antifungal, and anes-
thetic activities (Kuropatnicki et al., 2013). However, the chemical
composition of propolis is highly dependent on edaphoclimatic
conditions, and differences could influence biological activity and
the toxic responses to propolis (López et al., 2014).
In the last decade, many studies have investigated the biological
activities of red propolis, which is a variety found in northeastern
Brazil (Daugsch et al., 2008) and other developing countries such as
Cuba (Piccinelli et al., 2011) and Venezuela (Trusheva et al., 2004).
The botanical origin of red propolis from Alagoas (Northeast of Brazil)
was reported by Daugsch et al. (2008), as species of Leguminosae
family (Dalbergia ecastophyllum). Studies on hydroalcoholic extracts
n unreported bioactive compounds with unknown pharmacological
Corresponding author at: Tiradentes University, 300 Murilo Dantas Ave,
Farolândia, 49032-490 Aracaju, Sergipe, Brazil. Tel.: þ 55 79 3218 2190xR2615.
E-mail address: guetezanardo@yahoo.com.br (M. Zanardo Gomes).
of Brazilian red propolis have shown antioxidant (Frozza et al., 2013),
antitumor (Pinheiro et al., 2014), healing (Almeida et al., 2013), and
antiparasitic activities (Morsy et al., 2013), but its safety has not been
evaluated properly.
Red propolis has a specific chemical composition, and new
compounds never reported in other propolis varieties, such as
vestitol and neovestitol (Piccinelli et al., 2011; Bueno-Silva et al.,
2013), 3,4,20 ,30 -tetrahydroxychalcone, C-glycoside (Righi et al.,
2011), biochanin A, liquiritigenin, isoliquiritigenin, formononetin,
and medicarpin (Alencar et al., 2007; Piccinelli et al., 2011; Frozza
et al., 2013; López et al., 2014) have been detected in red propolis.
These chemical markers have antimicrobial (Daugsch et al., 2008),
cytotoxic against tumoral cells (Li et al., 2008), anti-inflammatory
(Bueno-Silva et al., 2013), anti-atherogenic, and anti-angiogenic
(Daleprane et al., 2012) properties.
Although commercial propolis extracts include compounds
recognized as safe substances and have popular health status,
these new propolis varieties represent an important source of
and toxicological actions (Czyżewska et al., 2014). In the present
study, we assessed the toxic effects of orally administering a

http://dx.doi.org/10.1016/j.jep.2015.05.009
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
 R.O. da Silva et al. / Journal of Ethnopharmacology 170 (2015) 66–71
hydroethanolic extract of red propolis (HERP), using international
protocols to study acute (OECD, 2001) and sub-acute toxicity
(OECD, 2008) in rats.
2. Materials and methods
2.1. Hydroalcoholic extract of red propolis (HERP)
Propolis was collected in Marechal Deodoro/Alagoas/Brazil
(S–91440 3600 and W–351520 300 ) in June 2013. The extraction was
performed using propolis samples (2 g) and 70% ethanol (25 mL)
at room temperature for 1 h in an ultrasound bath. After extrac-
tion, the mixture was filtered, and the solvent was evaporated to
produce HERP. The yield of extraction was 46% (w/w).
Chromatography analysis was conducted according to Alencar
et al. (2007) with minimal modifications. Briefly, a chromatograph
equipped with a DGU-20A3 degasser, a SIL-20A auto-sampler, two
LC-20AD pumps, an SPD-M20Avp photodiode array detector with
a CBM-20A interface, and a reverse-phase Phenomenex Luna C18
column (C-18, column size 4.6 mm  250 mm; particle size, 5 mm)
was used.
HERP was dissolved in methanol (high performance liquid
chromatography [HPLC] grade; Merck, Darmstadt, Germany)
(0.2 mg/ml) and filtered with a 0.45 mm filter (Millipore-HVHP,
Milford, MA, USA). A 20-ml aliquot of HERP was injected into the
HPLC system with a solvent flow rate of 1 mL/min. The column
was eluted with a linear gradient of acetic acid: water 1% v/v
(A) and methanol (B). The elution started with 40% B (10 min),
followed by 50–60% B (to 20 min), 65–70% B (to 35 min), and
finishing with 70–80% B (to 45 min).
The chromatograms were recorded at 280 nm, and formono-
netin, daidzein, and biochanin A authentic standards (Sigma Co.,
St. Louis, MO, USA) were used as biomarkers López et al., 2014.
2.2. Biological assay
2.2.1. Animals
Male and female Wistar rats (age, 8–12 weeks; males; 250–
350 g; females: 160–220 g) were used for acute and sub-acute
toxicology studies. Five rats were housed per polycarbonate cage
with free access to a normal diet and water. The rats were kept in a
temperature controlled room (22 73 1C), with a 12 h light/dark
cycle and relative humidity of o70%. The animals were acclimated
to the laboratory conditions for 5 days prior to the experiments. All
procedures were performed according to the Ethical Principles in
Animal Research adopted by the Brazilian College of Animal
Experimentation and approved by the Animals Ethics Committee
of Tiradentes University (Protocol no. 010913).
2.2.2. Acute toxicity study
The experiment was conducted according to the protocols
described in OECD Guideline 420 (OECD, 2001). The HERP extract
was dissolved in 5% (v/v) Tween 80 aqueous solution and admi-
nistered orally (300 mg/kg; HERP300) in a single dose. The control
group (CTR) received only the Tween 80 solution. The rats were
fasted overnight prior to dosing and 3 h after treatment. The
groups (HERP300 or CTRa) were comprised of female rats (n ¼ 5
each) and each received about 5 ml/kg (HERP 300 mg/mL or
solvent, respectively).
The general behavior of the rats (changes in skin, hair, eyes,
mucous membranes, and respiratory, circulatory, autonomic, and
central nervous systems, abnormal behavior, motor activity, tre-
mors, convulsion, salivation, diarrhea, lethargy, or sleep) was
monitored continuously during the first 24 h (0.25, 0.5, 1, 2, 4,
and 12 h) and daily until day 14 after dosing. The body weight of
67
the rats was measured on days 1, 7, and 14 (OECD 420, 2001). All
animals were euthanized by CO2 inhalation at the end of the
experiment, and their organs were excised and examined
macroscopically.
2.2.3. Sub-acute toxicity study
The experiment was conducted according to the protocols
described by OECD Guideline 407 (OECD, 2008). Wistar rats of
both sexes were assigned randomly to four groups (n ¼10/group:
five males and five females). The HERP extract was dissolved in 5%
(v/v) Tween 80 aqueous solution and administered orally daily for
28 days at single doses of 10 (HERP10), 100 (HERP100), and
200 mg/kg (HERP200) (volume  5 mL/kg), whereas the CTRsa
received only the solvent in the same volume.
The behavior and clinical signs of the rats, (same parameters
observed in acute study, including death) as well as the estrous
cycles (females) were observed daily. Body weight and water and
food intake were recorded weekly. Additional satellite groups of
four animals (two of each sex, control and highest dosage group–
HERP200) were prepared to observe the reversibility, persistence,
or delayed occurrence of toxic effects 14 days after the sub-acute
treatment.
Estrous cycles were monitored cytologically by vaginal lavage.
Vaginal samples were taken by flushing about 75 μL saline in and
out of the vagina and placing a drop of exudate on a slide. Vaginal
cytology was evaluated under light microscopy to determine the
estrous cycle stage based on the predominant cell type: proestrus:
presence of nucleated epithelial cells; estrus: cornified epithelial
cells; diestrus: leukocytes. Regular cycling was characterized by
short period, consisting of 2–3 days of diestrus (or metestrus),
1 day of proestrus, and 1–2 days of estrus.
On day 28, the animals were fasted 12 h prior to sampling of
blood collected via the superior mesenteric artery into non-
heparinized tubes for biochemical analyses and into EDTA tubes
for hematological analyses. This procedure was conducted under
anesthesia (100 mg kg  1 xylazine and 10 mg kg  1 ketamine, intra-
peritoneally). Full blood cell counts were determined using an
automatic hematology analyzer (BC5380 Mindray, Shenzhen,
PR China), and serum biochemistry tests were performed using a
clinical chemistry analyzer (Abbott Architect C8000; Abbott Med-
ical, Abbott Park, IL, USA), according to the manufacturer's instruc-
tions. Biochemical studies were carried out for liver function
(alkaline phosphatase – ALP, aspartate aminotransferase – AST,
and alanine aminotransferase – ALT), kidney function (urea, creati-
nine, and uric acid), and other biochemical parameters (glucose,
triglycerides, cholesterol, protein, sodium, potassium, and albumin).
After blood sampling, the animals were euthanized under CO2
inhalation, and the liver, kidneys, lungs, brain, spleen, heart, testes,
and ovaries were excised, weighed, and examined macroscopi-
cally. Relative organ weights were calculated as (organ/body
weight)  100. Organs, such as the liver, kidneys, and spleen, were
formalin-fixed, dehydrated, diaphanized, paraffin-embedded, and
5-mm thick sections of each sample were obtained and stained
with hematoxylin/eosin for histological examination. The micro-
scopic analysis of all tissue samples was carried out as a blind
study. The tissue samples were evaluated regarding the architec-
tural and cellular features of the parenchymal and stromal
components of the organs, as well as the presence of inflammatory
infiltrate, necrosis, or degenerative changes.
2.2.4. Statistics
All data are expressed as mean 7standard error. Student's
t-test was used for data generated in the acute study, and two-
way repeated-measures analysis of variance was used to deter-
mine differences between the groups in the sub-acute study.
68 R.O. da Silva et al. / Journal of Ethnopharmacology 170 (2015) 66–71
Tukey's post-hoc test was conducted for pairwise comparisons
when there was a significant overall difference between the
groups. All statistical analyses were performed using Prism ver.
5.0 (GraphPad Software, Inc., La Jolla, CA, USA). p-Values of o 0.05
were considered significant.
3. Results and discussion
3.1. Chemical composition as assessed by HPLC
HPLC analysis of HERP sample showed a complex chemical
composition with various peaks at different retention times
(Fig. 1). Peaks 1, 2, and 5 were identified as daidzein, formonone-
tin, and biochanin A, respectively. These compounds are red
propolis biomarkers (López et al., 2014). The main botanical
sources of red Brazilian propolis are at least two plant species,
and propolis composition varies regionally and possibly seasonally,
resulting in modifications in chemical composition. Unidentified
peaks showed retention time of 26.5 min with UV λ of 285 nm for
peak 3, 27.1 min with UV λ of 260, 286, 383 nm for peak 4. This
range of UV λ is compatible to flavonoids such as isoliquiritigenin
and liquiritigenin. Daugsch et al. (2008) studied red propolis from
the same region, and showed the presence of high amount of both
flavonoids. In addition, we observed the peak 6 (RT 39.7 min, UV λ
of 280, 330 nm), that could be attributed to prenylated benzophe-
nones. López et al. (2014) suggested that prenylated benzophe-
nones, such as guttiferone E, oblongifolin A, and xanthochymol,
could be major compounds in red propolis from Alagoas. Although
no benzophenone standards were available, they reported that the
main compound in the red propolis sample presented marker ion
m/z 601.35, suggesting the presence of these compounds.
Fig. 1. HPLC of HERP. (P1) daidzein, (P2) formononetin, (P3) RT 26.5 min, UV λ 285 nm, (P4) RT 27.1 min, UV λ of 260, 286, 383 nm, (P5) biochanin A, (P6) RT 39.7 min, UV λ of
280, 330 nm.
According to Alencar et al. (2007), no compound commonly
found in other types of propolis has been identified in red propolis,
demonstrating that chemical composition changes significantly. As
a result, this product may have peculiar biological and toxicologi-
cal profiles.
3.2. Acute toxicity study
No lethal effects or toxicity were observed after a single
administration of vehicle (CTRa) for the 14 days of the experiment.
Oral administration of HERP300 produced no lethal effects. How-
ever, 80% of the animals presented toxic signs until day 3 after
HERP administration. About 20% of the animals were sleepy with
decreased ambulation 15 min after administration. About 40%
were sleepy with decreased ambulation and wheezing after
30 min, and 20% were shivering. These symptoms indicate central
nervous system depressor effects. About 20% of the treated
animals developed diarrhea after 12 and 24 h and on day 3,
indicating an autonomous effect of HERP. No toxicity signs were
observed after day 3. According the OECD criteria, the occurrence
of toxicity signs in more than one animal and/or r one death
classified the product in the GHS 4 category, indicating a warning
(OECD, 2001; UNECE, 2011). The LD50 value for the oral dose of
HERP was 4300 mg/kg.
No significant differences in body weight gain were observed
between the HERP300 and CTRa groups after 7 and 14 days
(p o0.05). No altered growth or abnormal changes were detected
during the macroscopic analysis of the rat organs.
Araújo et al. (2011) observed little toxicity of a hydroalcoholic
extract of propolis from other Brazilian regions. Similar results
were reported by Mohammadzadeh et al. (2007), who found no
toxic effects after the ingestion of Iranian propolis. The altered
chemical composition of propolis, due to season or botanical
 R.O. da Silva et al. / Journal of Ethnopharmacology 170 (2015) 66–71 69

source, changes its toxicity profile (Teixeira et al., 2010). Red
propolis has novel compounds compared to those of commercial
propolis, which could be responsible for increasing its toxicity.
An acute dose study provides a guideline for selecting doses for
sub-acute dose study (OECD, 2001). Our red propolis acute toxicity
results indicate that a dose o 300 mg/kg could be used to assess
sub-acute toxicity. Thus, we chose 10, 100, and 200 mg/kg for
further studies.
2006). We demonstrated that red propolis is rich in isoflavones
with estrogenic effects, and that these substances have preference
to bind the ER-β receptor (Morito et al., 2001). In contrast,
estrogen receptor-alpha (ER-α) mediates the effects of estrogens
favoring loss of body weight. The significant increase in the body

3.3. Sub-acute toxicity study

No death was observed during the 28 days of treatment with

HERP at 10, 100, or 200 mg/kg. However, some toxic signs were
observed, such as diarrhea (20% of animals treated with vehicle,
30% using HERP 10, 60% using HERP 100, and 50% using HERP
200), wheezing (10% using HERP 100), and decreased ambulation
(20% using HERP 100). Wheezing was observed on day 1 and
decreased ambulation was detected in males on days 5 and 6.
These signs did not persist after this period.
The sub-acute oral administration of HERP (10, 100, and
200 mg/kg daily for 28 days) resulted in insignificant changes in
food and water intake (Fig. 2) in the treated rats compared with
those in the control. We observed significant changes in food
intake (males: weeks 1 and 3 in HERP 200 and females: week 3 in
HERP 100; p o0.05) and water intake (males: week 1 in HERP 200
and females: week 3 in HERP 100). However, the increase in body
weight in the females treated with HERP 200 was significant
compared with that in the control group during the entire
experiment (p o0.05) (Fig. 3). This result may be related to the
estrogen-mimetic action of the phytoestrogens present in red
propolis. Fig. 3. Body weight gain by week of rats, female (A) and male (B), of control group
The role of endogenous estrogens in regulating body weight in and treated with hydroalcoholic extract of red propolis (HERP) at doses of 10, 100
and 200 mg/kg in the sub-acute toxicity study. Values expressed in percentage
females is well supported in the literature and demonstrates their
compared to the first day of treatment and the bars indicate the standard error of
interactions with different types of receptors. Estrogen binding to mean. Significant in relation to control at npo 0.05; nnp o0.01, nnnpo 0.001. ANOVA
estrogen receptor-beta (ER-β) does not affect body weight (Roesch, two way (Tukey test).

Fig. 2. Weekly consumption of water (mL/100 g body weigh) and food (g/100 g body weight) of animals (male and female) of control group and treated with hydroalcoholic
extract of red propolis (HERP) at doses of 10, 100 and 200 mg/kg in the sub-acute toxicity study. Significant in relation to control at np o0.05, nnnp o 0.001. ANOVA two way
(Tukey test).
70 R.O. da Silva et al. / Journal of Ethnopharmacology 170 (2015) 66–71

masses of the female rats treated with HERP200 can be explained
by the negative feedback effect of decreasing endogenous estrogen
level when the animals are exposed to high isoflavone concentra-
tions (Lu et al., 1996). Thus, the absence of the endogenous
estrogen modulating ER-α may have increased body mass, similar
to that occurring at menopause or after ovariectomy (Roesch,
2006).
All female rats in the CTRsa group showed a normal sequence of
changes on the vaginal smear. However, the order of appearance of
the various phases and the duration of the estrus cycle changed in
40% of HERP10 and in 100% of HERP100 and HERP200-treated rats
during a 28-day period.
The rat estrous cycle is characterized by an increase in estro-
gens on the second day of diestrus, resulting in increased luteiniz-
ing hormone from the pituitary, followed by a short luteal phase
and estrus (OECD, 2009; Andersson et al., 2013). Thus, disrupting
the estrus cycle with HERP may have been associated with some
hormonal disturbances in the hypothalamo-pituitary–gonadal
axis. Moreover, disruptions in the estrus cycle were more evident
in the first and second weeks of treatment with HERP100 (80% of
rats) and in the third and fourth weeks of treatment with
HERP200 (80%), which was associated with the changes in body
weight.
We observed a significant increase in spleen weight in the
HERP100 group compared to that in the CTR group (po0.001)
(Table 1). Changes in the mass of lymphoid organs (spleen and
thymus) should be analyzed together with relevant histopathological
findings due to weight variations in these organs (Sellers
et al., 2007). There were no changes in spleen tissue (Figure S1,
Supporting information), demonstrating that this event was irrele-
vant to the spleen. The other organs showed no differences (Table 1).
The male results revealed a significant increase in lung weight
(p o0.01) in the HERP100 group. According to the Society for
Toxicological Pathology, lung weight is an important parameter to
evaluate inhalation medicine. An assessment of lung weight in an
oral administration study adds little value to a microscopic
evaluation and should be optional (Sellers et al., 2007). However,
we found a decrease in testicular mass (p o0.01) in the HERP200
compared with that in the CTR group (Table 1). According to Bae
et al. (2012), daidzein, formononetin, and biochanin inhibit
5α-reductase, which converts testosterone to dihydrotestosterone
(more potent androgen). Reducing the level of this hormone
promotes androgenic diseases, such as benign prostatic hyperpla-
sia. Caceres et al. (2014) showed that orally administering iso-
flavones to rats decreases androgen secretion. This inhibition of
hormones may have decreased the testicular weight in our study.
The hematological parameters of the female rats revealed a
significant decrease in mean corpuscular volume (MCV) in the
HERP100 (po0.05) and HERP200 (po0.001) groups and increased
mean corpuscular hemoglobin concentration (MCHC) in the HERP200
(po0.01) compared with that in the CTRsa group (Table S1, Supporting
information).
A significant increase in red blood cell (RBC) (p o0.05) and
hemoglobin values were observed in males treated with HERP10
and HERP100 (p o0.05). The HERP100 group also had decreased
MCHC values (p o0.05). The HERP200 treatment decreased the
number of eosinophils (p o0.05) and increased the number of
monocytes (po 0.01) (Table S1, Supporting information).
Despite observing some significant changes in the hematolo-
gical parameters, most values remained within normal ranges
( Lima et al., 2014). However, RBC and hemoglobin values in males
(HERP10 and HERP100) were outside the normal reference values
(Lima et al., 2014).
The HERP10 treatment significantly increased urea level compared
with that in the CTR group (Table S2, Supporting information).
HERP100 animals showed high levels of ALP, and the HERP200 group
presented increase sodium and total cholesterol values. Despite the
significant differences, these values remained within acceptable limits
(Lima et al., 2014). We observed a significant reduction in triglycerides
and increases in total protein, sodium, ALP, creatinine, total cholesterol,
ALT, and albumin in HERP200 male animals compared to those in the
CTR group (Table S2, Supporting information). However, these values
were also within normal ranges (Lima et al., 2014).
Although the hematological and biochemical results remained
within the reference values (Lima et al., 2014), the possibility of
toxicity after administering HERP daily for 28 days was considered,
because all results together indicated tendencies for changes in
relevant clinical parameters.
In this study, we observed an increase in ALT values in male rats
that suggest liver necrosis or changes in blood capillary membrane
permeability. An increase in total protein and a decrease in triglycer-
ides occurred in male animals. These data are similar to those in the
red propolis study by Morsy et al. (2013). They used a similar
biochemical parameter profile in a sheep model.
The chemical composition and dosage may be strongly related
to the potential toxic effects of HERP. For example, the compound
genistein, the metabolized product from biochanin A, was toxic
when acutely administered upper to 500 mg/Kg (i.p.) in mice. It
elevated ALT, AST, and ALP levels, induced degeneration of liver
tissue, and produced oxidative stress with lipid peroxidation and
reduction in total glutathione levels (Singh et al., 2014). In vitro
studies showed that oxidative metabolism of daidzein promotes

Table 1
Effects of hydroalcoholic extract of red propolis (HERP – 10, 100 and 200 mg/kg) by oral route on relative organ weight (organ weight/body weight%) in male and female
Wistar rats treated for 28 consecutive days.

Organ Females Males

Control HERP 10 HERP 100 HERP 200 Control HERP 10 HERP 100 HERP 200

Brain 0.83 7 0.02 0.82 7 0.01 0.86 70.02 0.82 70.03 0.577 0.01 0.58 7 0.03 0.59 7 0.02 0.53 7 0.01
Liver 3.26 7 0.08 3.517 0.16 3.94 70.30 3.26 70.01 2.747 0.08 2.717 0.06 2.79 7 0.16 2.83 7 0.12
Kidneys 0.86 7 0.02 0.86 7 0.03 0.91 70.04 0.91 70.07 0.83 7 0.01 0.82 7 0.02 0.85 7 0.04 0.79 7 0.3
Lungs 0.447 0.01 0.46 7 0.02 0.47 70.02 0.49 70.02 0.357 0.03 0.447 0.03 0.50 7 0.03** 0.417 0.03
Heart 0.39 7 0.01 0.447 0.2 0.42 70.02 0.42 70.02 0.37 7 0.01 0.36 7 0.01 0.357 0.01 0.357 0.02
Stomach 0.92 7 0.06 0.87 7 0.08 1.02 70.10 1.26 70.04 0.727 0.08 0.56 7 0.02 0.747 0.10 0.777 0.05
Spleen 0.177 0.01 0.177 0.00 0.2470.01*** 0.18 70.01 0.137 0.01 0.157 0.01 0.137 0.00 0.137 0.00
Ovaries 0.03 7 0.00 0.03 7 0.00 0.03 70.00 0.02 70.00 – – – –
Testicles – – – – 0.447 0.01 0.477 0.01 0.46 7 0.01 0.38 7 0.01**

Values represent the mean7 SEM (n¼ 5/group). ANOVA one way (Tukey test).
nn
Significant in relation to control at p o 0.01
nnn
Significant in relation to control at po 0.001.
 R.O. da Silva et al. / Journal of Ethnopharmacology 170 (2015) 66–71
the formation of hydroxylated metabolites that were genotoxic to
human sperm cells and lymphocytes (Baechler et al., 2014). Also,
biochanin A was toxic to human endothelial cells in a dose-
dependent manner (Paulo and Mota-Filipe, 2006). Although dif-
ferences exist regarding the minimal cytotoxic concentrations of
isoflavones reachable in human plasma to the healthy cells in
relation to the cultured cells, the data above suggest caution on
administrating high levels of these compounds.
No signs of delayed toxicity due to the HERP treatment
occurred in the satellite group. Histological evaluations of the
liver, kidneys, and spleen in the CTRsa and HERP200 groups
showed no changes (Figure S1, Supporting information).
In conclusion, administering HERP to rats resulted in higher
toxicity than that observed when administering other propolis
types in acute and sub-acute studies, indicating that chemical
composition interferes significantly in the biological response. Our
results show that the LD50 was higher than 300 mg/kg HERP. The
classification based on the OECD Guidelines suggests a warning for
sub-acute administration of doses higher than 100 mg/kg.
Acknowledgments
The authors thank FAPITEC/SE and CNPQ (grants number PPP
04/2011 and PRONEM 10/2011) for financial support and bee-
keepers for providing the propolis samples.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2015.05.009.
References
Alencar, S.M., Oldoni, T.L.C., Castro, M.L., Cabral, I.S.R., Costa-neto, C.M., Cury, J.A.,
Rosalen, P.L., Ikegaki, M., 2007. Chemical composition and biological activity of
a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113, 278–283.
Almeida, E.B., Cardoso, J.C., Lima, A.K., Oliveira, N.L., Pontes-Filho, N.T., Lima, S.O.,
Souza, I.C.L., Albuquerque-júnior, R.L.C., 2013. The incorporation of Brazilian
propolis into collagen-based dressing films improves dermal burn healing.
J. Ethnopharmacol. 147, 419–425.
Andersson, H., Rehm, S., Stanislaus, D., Wood, C.E., 2013. Scientific and regulatory
policy committee (SRPC) paper: assessment of circulating hormones in non-
clinical toxicity studies III female reproductive hormones. Toxicol. Pathol. 41,
921–934.
Araújo, M.J., Mattar, N.S., Reis, A.S., Serra, I.C., Fialho, E.M., Assunção, A.K., Dutra, R.
P., Nogueira, A.M., Libério, S.A., Guerra, R.N., Lopes, A.S., Ribeiro, M.N., Nasci-
mento, F.R., 2011. Pharmacognostic and acute toxicological evaluation of
Scaptotrigona aff. postica propolis extract in pre-clinical assays. Nat Prod Res.
25, 1037–1046.
Bae, M., Woo, M., Kusuma, I.W., Arung, E.T., Yang, C.H., Kim, Y., 2012. Inhibitory
effects of isoflavonoids on rat prostate testosterone 5a-reductase. J. Acupunct.
Meridian Stud. 5, 319–322.
Baechler, S.A., Schroeter, A., Walker, J., Aichinger, G., Marko, D., 2014. Oxidative
metabolism enhances the cytotoxic and genotoxic properties of the soy
isoflavone daidzein. Mol. Nutr. Food Res. 58, 1269–1281.
Bueno-Silva, B., Alencar, S.M., Koo, H., Ikegaki, M., Silva, G.V.J., Napimoga, M.H.,
Rosalen, P.L., 2013. Anti-inflammatory and antimicrobial evaluation of neoves-
titol and vestitol isolated from Brazilian red propolis. J. Agric. Food Chem. 61,
4546–4550.
Caceres, S., Silvan, G., Martinez-Fernandez, L., Illera, M.J., Millan, P., Monsalve, B.,
Peña, L., Illera, J.C., 2014. The effects of isoflavones on androgens and
glucocorticoids during puberty on male Wistar rats. Reprod. Domest. Anim.
49, 611–617.
Czyżewska, U., Konończuk, J., Teul, J., Drągowski, P., Pawlak-Morka, R., Surażyński, A.,
Miltyk, W., 2014. Verification of chemical composition of commercially
71
available propolis extracts by gas chromatography-mass spectrometry analysis.
J. Med. Food 00, 1–8.
Daleprane, J.B., Freitas, V.S., Pacheco, A., Rudnicki, M., Faine, L.A., Dörr, F.A., Ikegaki,
M., Salazar, L.A., Ong, T.P., Abdalla, D.S.P., 2012. Anti-atherogenic and anti-
angiogenic activities of polyphenols from própolis. J. Nutr. Biochem. 23,
557–566.
Daugsch, A., Moraes, C.S., Fort, P., Park, Y.K., 2008. Brazilian red propolis—chemical
composition and botanical origin. Evid. Based Complement. Altern. Med. 5,
435–441.
Frozza, C.O.S., Garcia, C.S.C., Gambato, G., Souza, M.D.O., Salvador, M., Moura, S.,
Padilha, F.F., Seixas, F.K., Collares, T., Borsuk, S., Dellagostin, O.A., Henriques, J.A.
P., Roesch-Ely, M., 2013. Chemical characterization, antioxidant and cytotoxic
activities of Brazilian red propolis. Food Chem. Toxicol. 52, 137–142.
Kuropatnicki, A.K., Szliszka, E., Krol, W., 2013. Historical Aspects of Propolis
Research in Modern. Evid. Based Complement. Altern. Med. 2013, 1–11.
Li, F., Awale, S., Tezuka, Y., Kadota, S., 2008. Cytotoxic constituents from Brazilian
red propolis and their structure–activity relationship. Bioorg. Med. Chem. 16,
5434–5440.
Lima, C.M., Lima, A.K., Melo, M.G.D., Dória, G.A.A., Leite, B.L.S., Serafini, M.R.,
Albuquerque-Júnior, R.L.C., Araújo, A.A.S., 2014. Valores de referência hemato-
lógicos e bioquímicos de ratos (Rattus novergicus linhagem Wistar) prove-
nientes do biotério da Universidade Tiradentes. Sci. Plena 10, 034601.
López, B.G.C., Schmidt, E.M., Eberlin, M.N., Sawaya, A.C.H.F., 2014. Phytochemical
markers of different types of red propolis. Food Chem. 146, 174–180.
Lu, L.W., Anderson, K.E., Grady, J.J., Nagamani, M., 1996. Effects of soya consumption
for one month on steroid hormones in premenopausal women: implications for
breast cancer risk reduction. Cancer Epidemiol. Biomark. Prev. 5, 63–70.
Mohammadzadeh, S., Shariatpanahi, M., Hamedi, M., Ahmadkhaniha, R., Samadi, N.,
Ostad, S.N., 2007. Chemical composition, oral toxicity and antimicrobial activity
of Iranian propolis. Food Chem. 103, 1097–1103.
Morito, K., Hirose, T., Kinjo, J., Hirakawa, T., Okawa, M., Nohara, T., Ogawa, S., Inoue,
S., Muramatsu, M., Masamune, Y., 2001. Interaction of phytoestrogens with
estrogen receptors α and β. Biol. Pharm. Bull. 24, 351–356.
Morsy, A.S., Abdalla, A.L., Soltan, Y.A., Sallam, S.M.A., El-Azrak, K.E.M., Louvandini,
H., Alencar, S.M., 2013. Effect of Brazilian red propolis administration on
hematological, biochemical variables and parasitic response of Santa Inês ewes
during and after flushing period. Trop. Anim. Health Prod. 45, 1609–1618.
OECD, 2001. Guideline for testing of chemicals. Acute Oral Toxicity – Fixed Dose
Procedure. Adopted 17th December 2001.
OECD, 2008. Guideline for testing of chemicals. Repeated dose 28-day oral toxicity
study in rodents. Adopted 3 October 2008.
OECD, 2009. Guidance document for histologic evaluation of endocrine and
reproductive tests in rodents. 28 July 2009.
Paulo, A., Mota-Filipe, H., 2006. Effects of some natural 5-hydroxy-isoflavones on
cultured human endothelial cells in presence and absence of hydrogen
peroxide. J. Pharm. Pharmacol. 58, 101–105.
Piccinelli, A.L., Lotti, C., Campone, L., Cuesta-Rubio, O., Fernandez, M.C., Rastrelli, L.,
2011. Cuban and Brazilian red propolis: botanical origin and comparative
analysis by high-performance liquid chromatography-photodiode array detec-
tion/electrospray ionization tandem mass spectrometry. J. Agric. Food Chem.
59, 6484–6491.
Pinheiro, K.S., Ribeiro, D.R., Alves, A.V.F., Pereira Filho, R.N., Oliveira, C.R., Lima, S.O.,
Reis, F.P., Cardoso, J.C., Albuquerque-Júnior, R.L.C., 2014. Modulatory activity of
brazilian red propolis on chemically induced dermal carcinogenesis. Acta Cir.
Bras. 29, 111–117.
Righi, A.A., Alves, T.R., Negri, G., Marques, L.M., Breyer, H., Salatino, A., 2011.
Brazilian red propolis: unreported substances, antioxidant and antimicrobial
activities. J. Sci. Food Agric. 91, 2363–2370.
Roesch, D.M., 2006. Effects of selective estrogen receptor agonists on food intake
and body weight gain in rats. Physiol. Behav. 87, 39–44.
Sellers, R.S., Morton, D., Michael, B., Roome, N., Johnson, J.K., Yano, B.L., Perry, R.,
Schafer, K., 2007. Society of toxicologic pathology position paper: organ weight
recommendations for toxicology studies. Toxicol. Pathol. 35, 751–755.
Singh, P., Sharma, S., Rath, S.K., 2014. Genistein induces deleterious effects during
its acute exposure in Swiss mice. BioMed Res. Int. 2014, 619617.
Teixeira, E.W., Message, D., Negri, G., Salatino, A., Stringheta, P.C., 2010. Seasonal
variation, chemical composition and antioxidant activity of Brazilian propolis
samples. Evid. Based Complement. Altern. Med. 7, 307–315.
Trusheva, B., Popova, M., Naydenski, H., Tsvetkova, I., Rodriguez, J.G., Bankova, V.,
2004. New polyisoprenylated benzophenones from Venezuelan propolis. Fito-
terapia 75, 683–689.
UNECE – United Nations Economic Commission for Europe. 2011. Globally Harmo-
nized System of Classification and Labelling of Chemicals (GHS). 〈http://www.
unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev04/English/ST-SG-A
C10-30-Rev4e.pdf〉 (accessed 08.09.13.).