Effect of propolis on the healing of ethanol- and acetic acid chronic gastric ulcer in rats

Abstract

This study examined the effect of propolis on the healing of ethanol- and acetic acid-induced acute and chronic
gastric ulcers. In addition, the effect of anti-ulcer drugs like cimetidine and maalox on ulcer healing was investigated.
In a rat model, ethanol-induced gastric ulcers were healed after single and repeated administration of
drugs. Administration of propolis promoted ulcer healing, i.e., ulcers were healed 7 days after ulcer induction,
3 days earlier than the control. Administration of cimetidine at a higher dose further promoted ulcer healing.
After administration of propolis, the surface of acetic acid-induced gastric ulcers and the surface of duodenal
ulcers were diminished by more than 50%. These results indicate that propolis favours the healing of acetic
acid-induced gastric and duodenal ulcers.

Full text

ACTA BIOLOGICA CRACOVIENSIA Series Zoologia 52: 37–43, 2010
PL ISSN 0001-530X © Polish Academy of Sciences, Cracow 2010
1 e-mail: mibrow@cyf-kr.edu.pl
INTRODUCTION
Constantly raising interest in the therapeutic
properties of propolis and continuous growth in
the number of patients receiving propolis treat-
ment, which has been noted over recent years,
collide with the fact that there is a lack of de-
tailed data on its pharmacological actions (Ag a
et al., 1994; Ba n s k o t a et al., 2000; Ca s t a l d o
and Ca p a s s o , 2002; Be l o s t o t s k i j et al., 2009;).
Propolis (beeswax) is a mixture containing dif-
ferent compounds such as avonoids, phenolic
and amino acids, and trace elements such as zinc
(Ma r c u c c i , 1995; Bu r d o c k , 1998; Ba n s k o t a et al.,
2001; Mi d o r i k a w a et al., 2001; Ha v s t e e n , 2002).
The presence of zinc can explain the potential
anti-inammatory activity of propolis. As shown
in the literature, propolis is effective in the
therapy of many diseases (Sc h e l l e r et al., 1980;
Kr a s n o d ę b s k i et al., 1986; Ha r t w i c h et al., 2002).
Also the anti-cancer activity of propolis has been
reported (Ci z m a r i k and La h i t o v a , 1998; Li n et al.,
1999; Lu o et al., 2001; So n g et al., 2002). Various
propolis preparations have long been used in folk
medicine for treatment of an array of diseases, es-
pecially those accompanied by purulent process-
Ef f e c t of Pr o p o l i s o n t h e He a l i n g o f Et h a n o l - a n d Ac e t i c
Ac i d -in d u c e d Ch r o n i c Ga s t r i c Ul c e r i n Ra t s
ry s z a r d Cz a r n e C k i , Ta d e u s z Li b r o w s k i 1
Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University,
Medyczna 9, 30-688 Cracov, Poland
Accepted November 24, 2010
This study examined the effect of propolis on the healing of ethanol- and acetic acid-induced acute and chronic
gastric ulcers. In addition, the effect of anti-ulcer drugs like cimetidine and maalox on ulcer healing was inves-
tigated. In a rat model, ethanol-induced gastric ulcers were healed after single and repeated administration of
drugs. Administration of propolis promoted ulcer healing, i.e., ulcers were healed 7 days after ulcer induction,
3 days earlier than the control. Administration of cimetidine at a higher dose further promoted ulcer healing.
After administration of propolis, the surface of acetic acid-induced gastric ulcers and the surface of duodenal
ulcers were diminished by more than 50%. These results indicate that propolis favours the healing of acetic
acid-induced gastric and duodenal ulcers.
Key words: Propolis, gastric ulcer, duodenal ulcer, anti-ulcer agents

38 Czarnecki and Librowski
es. In many research centres, studies on propolis
chemical composition and main constituents with
antibacterial activity have been conducted for
many years (Ag a et al., 1994; Ch o p r a et al., 1995;
Ka j u m g i e v et al., 1999; Ke s k i n et al., 2001; Ko o
et al., 2002; Sa n t o s et al., 2002; Bo y a n o v a et al.,
2003; Du a r t e et al., 2003; St e p a n i v i c et al., 2003).
So far, the mechanism of the antibacterial action
of propolis has not been elucidated in detail. It is
believed to be complex and to involve synergistic
interaction of different compounds, including a-
vonoids, hydroxyacids and sesquiterpenes (Ha v s -
t e e n , 2002; Pi e t t a et al., 2002).
Data on such propolis properties as its effect
on arterial blood pressure, central nervous sys-
tem, smooth muscles, and its cholepoietic or tis-
sue-regenerating actions are incomplete and of-
ten contradictory (Ba z o et al, 2002; Gr e g o r y et al.,
2002; Pa u l i n o et al., 2002; Pa u l i n o et al., 2003). It
seems to be due to the fact that the explanation
of both biological and pharmacological activities
of propolis will require simultaneous resolution of
many theoretical and practical problems. At pre-
sent, difculties are encountered in interpreting
therapeutic effects of propolis, which results from
the hitherto unexplained mechanisms of its action
in many diseases and sometimes even divergent
pathogenetic mechanisms (Su c h y et al., 1974).
The main goal of the present study was to
evaluate the protective action of condensed etha-
nol extract of propolis in the model of experimen-
tal peptic ulcer in rats.
MATERIAL AND METHODS
Pharmacological studies were conducted using
condensed propolis extract suspended in 1%
methyl cellulose. Propolis was obtained from
apiaries located in ecologically protected areas in
southern Poland. Propolis was delivered to the De-
partment of Pharmacodynamics directly from the
apiaries remaining under veterinary super vision.
Animals
The animals used for the tests came from the
Ani mal Breeding Farm of the Faculty of Phar-
macy, Jagiellonian University, Medical College.
The ani mals had free access to standard pellet
diet and water, and were used after a minimum
of 3 days of acclimation to the housing condi-
tions. Experimental gastric and duodenal ulcers
were induced in white Wistar male rats, weigh-
ing 160–180 g, kept in standard cages in a room,
at a temperature of 20–24oC, with 12 h/12h light/
dark cycle. The rats were fed standard granulated
LSM feed. Each experimental group consisted of
8–12 animals and all the animals were used only
once. The experiments were performed between
8 a.m. and 3 p.m. The animals were housed in
animal compartments under constant supervi-
sion of the University Senate Bioethics Commis-
sion for Care of Laboratory Animals. The animals
were starved for 24 hours before the experiments
but had free access to water. Experimental gas-
tric and duodenal ulcers were induced using two
methods.
Acute experimental gastric ulcer induced
by ethanol administration in rats
Gastric mucosa damage was evoked by the ad-
ministration of 1 ml/rat of 99.8% ethanol p.o. 1
hour before sacrice according to the method
drescribed and modied by Okabe and Takagi
(Ta k a g i et al., 1969; Ok a b e et al., 1993; Ok a b e
et al., 1997). Stomach and duodenum were exa-
mined macroscopically and microscopically. Mu-
cosal lesions, i.e. erosions and ulcerations were
measured planimetrically and presented as
means. Both the number and extent of lesions
were reported. The control group was composed
of animals receiving only the ulcerative agent.
Propolis at a dose of 10 mg/kg was adminis-
tered p.o. either as a single dose or multiple doses
(twice a day for 10 days). The control group rats
were administered 0.1 ml/10 g of body weight of
1 % methyl cellulose in single or multiple doses.
The control drug for single administrations was
cimetidine (Tagamet – Smith, Kline & French, F)
at a dose of 75 mg/kg, suspended in 0.9 % NaCl,
which was given p.o. in a volume of 0.1 ml/100 g,
and algeldrate (Maalox – Rore, CDN) adminis-
tered in a volume of 0.1 ml/100 g of body weight.
All the studied compounds were administered
prophylactically 60 minutes before the ulcerative
agent.

Inuences of propolis on experimental ulcers 39
Chronic experimental gastric and duodenal
ulcer induced by acetic acid in rats
Model, chronic gastric ulcer was evoked with gla-
cial acetic acid applied in a volume of 40 ml to
an area of 13.85 mm2 of the body of the stomach
over 20 seconds (Ta k a g i et al., 1969; Ok a b e et al.,
1993; Ok a b e et al., 1997). Experimental duodenal
ulcer was induced with 75 % acetic acid adminis-
tered onto the duodenal mucosa 5 mm below the
pylorus over 10 seconds. The animals were under
amobarbital anaesthesia (Amobarbital sodium
– Eli, Lilly & Co.). The ulcer area was measured
planimetrically at three intervals: on the day of
ulcer induction, i.e. day “0”, and on the 7th and
10th days of the experiment. Propolis at a dose of
10 mg/kg p.o. and methyl cellulose in a volume
of 0.1 ml/100 g of body weight p.o. were adminis-
tered twice a day for 7 or 10 consecutive days.
Statistical analysis
The antiulcerative effect was expressed as a per-
centage decrease in the average area of gastric
or duodenal ulcer in comparison with the control
group. Student’s t-test was used to determine the
signicance of differences between the mean val-
ues for the control and treatment groups. Diffe -
rences were considered signicant when p < 0.05.
RESULTS
Ethyl alcohol (99.8%) caused damage to the gas-
tric mucosa in 100% of the studied animals. In
the control group, the average lesion area was
197.02 mm2 per rat, while the number of mu-
cosal erosions averaged 25.8 per rat. Propolis de-
creased the average lesion area by about 66% in
comparison with the control group (p < 0.001).
The number of ulcerations was also reduced by
approximately 40% in comparison with the con-
trol group (p < 0.01). In animals that were admi-
nistered propolis, weak gastric mucosa oedema
and less numerous extravasations, supercially
penetrating the mucosa and the submucosal
layer, were observed. No changes were observed
in the duodenal mucosa. Methyl cellulose de-
creased the ulcer area by about 45% but it did
not inuence the number of ulcerations in a sta-
tistically signicant way. Microscopic exami-
nations did not indicate that methyl cellulose
prevented congestion, blood extravasations and
haemorrhagic erosions of the gastric mucosa.
Cimetidine administered at a dose of 75 mg/kg
p.o. signicantly decreased both the ulcer area
and the number of ulcerations in the stomach
(p < 0.001) but it did not show protective action
on the duodenal mucosa. Algeldrate reduced the
average duodenal ulcer area by approximately
50% and signicantly diminished the average
number of gastric ulcerations (p < 0.01) (Table 1).
Multiple administration of propolis decreased
the average ulcer area by about 82% (p < .0.001)
but had little inuence on the number of ulcer-
ations. Microscopic examination gave only trace
indications of changes in the gastric mucosa in
the form of tiny erosions and extravasations. The
duodenal mucosa was only slightly congested and
had a clearly visible and well-preserved villus
structure. Although methyl cellulose statistically
signicantly reduced the average ulcer area by
about 60% (p < 0.01), it increased the number of
ulcerations by 27% in comparison with the con-
trol. The picture of changes was similar to that
for the control group (Table 2).
After the induction of chronic experimental
gastric and duodenal ulcer with acetic acid, the
ulcer area averaged 13.85 mm2 on the day of
ulcer induction (day “0”). On the 7th and 10th
days, peptic ulcers were observed in all the con-
trol animals. The average ulcer area decreased
by about 60% in the stomach, and by approxi-
mately 60% and 84% on the 7th and 10th days,
respectively, in the duodenum, in comparison
with respective values on day “0”. In the ani-
mals treated with propolis, the average ulcer
area decreased by 76% and 78% in the stomach
and duodenum, respectively, on the 7th day in
comparison with the value obtained on day “0”
(p < 0.05). In the animals that were adminis-
tered methyl cellulose the average ulcer area
was reduced by 75% (p < 0.05) in the stomach
and by 79% (p < 0.05) in the duodenum on the
7th observation day in comparison with day “0”.
On the 10th day of the experiment no protective
methyl cellulose action, either in the stomach
or in the duodenum, was observed. The average
ulcer areas were similar to those for the control
group (Table 3).

40 Czarnecki and Librowski
TABLE 1. The effect of oral administration of propolis, methylcellulose (MCL-25), cimetidine and maalox on acute experimental
gastric ulcers generated with ethanol in rats.
Single administration. Route = p.o.; N = 8–12
Compound Dose
The average surface
of gastric ulcers
(mm2)
% of
inhibition
The average number
of gastric ulcers % of inhibition
Control
Propolis
MCL-25
Cimetidine
Maalox
---------
10 (mg/kg)
1 (ml/kg)
75 (mg/kg)
1 (ml/kg)
197.0 ± 22.
65.91 ± 16.5c
107.3 ± 24.9
57.8 ± 18.7c
97.4± 25.7
---------
33.45
54.46
29.34
49.44
25.8 ± 2.7
15.5 ± 1.4b
22.6 ± 2.7a
12.8 ± 1.7c
18.7 ± 3.3b
-----------
60.07
87.59
49.61
72.48
Signicant difference (Student’s t-test) compared to the vehicle-treated group: ap < 0.02; bp < 0.01; cp < 0.001. N = number of
animals
TABLE 2. The effect of oral administration of propolis and methylcellulose (MCL-25) on experimental gastric ulcers generated
with ethanol in rats.
Repeated administration. Route = p.o.; N = 8–12
Compound Dose
The average surface
of gastric ulcers
(mm2)
% of
inhibition
The average
number of gastric
ulcers
% of inhibition
Control
Propolis
MCL-25
---------
10 (mg/kg)
1 (ml/kg)
197.0 ± 22.2
35.9 ± 10.4c
78.3 ± 27.2b
---------
18.22
39.74
25.8 ± 2.7
22.6 ± 5.0
32.8 ± 7.0
-----------
87.56
127.13
Signicant difference (Student’s t-test) compared to the vehicle-treated group: ap < 0.02; bp < 0.01; cp<0.001. N = number of
animals
TABLE 3. The effect of oral administration of propolis and methylcellulose (MCL-25) on decreasing chronic experimental
gastric and duodenal ulcers generated with acetic acid in rats.
Compound
Day of observation
0 7 10
N
gastric
ulcers
duodenal
ulcers N
gastric
ulcers
duodenal
ulcers N
gastric
ulcers
duodenal
ulcers
(mm2) % (mm2) % (mm2) % (mm2) % (mm2) % (mm2) %
Control 12 13.8 100 13.8 100 11 5.7 ± 0.9 41.3 5.4 ± 1.1 39.1 11 5.7 ± 1.4 41.3 2.2 ± 0.4 15.9
Propolis 12 13.8 100 13.8 100 9 3.2 ± 0.6 23.2 3.1 ± 0.7 22.5 9 3.2 ± 0.7 23.2 1.1 ± 0.4 7.9
MCL-25 12 13.8 100 13.8 100 8 3.4 ± 1.4 24.6 2.9 ± 0.5 21.0 12 5.1 ± 0.8 36.9 1.9 ± 0.5 13.7
N = number of animals

Inuences of propolis on experimental ulcers 41
DISSCUSSION
According to the commonly accepted research
methodology, pharmacological assessment of the
antiulcerative action of new substances of natural
origin involves the determination of drug capaci-
ty to prevent development of experimental ulcers
induced by various procedures, typically, in rats.
The antiulcerative activity of propolis depends
not only on its pharmacological activity but also
on the sensitivity of animals to ulcerative agents.
In consequence, many methodological and theo-
retical problems arise which require resolving.
One of such problems is caused by a wide scatter
of results which creates signicant difculties in
assessing the antiulcerative activity of a prepa-
ration, especially if new substances of natural
origin are investigated.
Taking into consideration all the above-
-mentioned remarks, the present studies were
undertaken with the main goal to assess the
antiulcerative activity of propolis. The detailed
exa mination of this natural product was justi-
ed by the results of preliminary experiments
which showed its inhibitory effect on the deve-
lopment of experimental gastric ulcers in rats.
Further substantiation of this research, deser-
ving attention and interest, is provided by the
fact that its antiulcerative activity claimed in
folk medicine has not been satisfactory clari-
ed yet. The results of the studies on protective
properties of propolis, presented in this paper,
justify the conclusion that under both dosage
regimens (single and multiple administration)
it exhibits the activity characteristic of antiul-
cerative agents on experimental peptic ulcers
induced by 99.8 % ethanol in rats.
The experiments on the protective action of
propolis on chronic peptic ulcers induced by ace-
tic acid administration in rats also appear to in-
dicate that this substance exerts actions charac-
teristic of antiulcerative agents. Obviously, it can
be a matter of further discussion whether it is the
cytoprotective or receptor action as both groups
of drugs evoke similar changes in experiments on
animals. The basic difculty in search for a mecha-
nism of propolis action is lack of pharmacological
assessment of its adrenolytic, cholinolytic, anti-
histamine, spasmolytic and anxiolytic properties.
Therefore, the postulation that any conception of
the antiulcerative action of propolis, even that
simplied, is the only one would be a signicant
oversimplication when confronted with very
complex pathogenesis of gastric ulcer, depending
on the ulcerative agent besides.
The literature data (Ta k a g i and Ok a b e , 1969;
Ok a b e et al., 1977; Ki d e r m a n et al., 2001; Be-
l o s t o t s k i j et al., 2009) indicate that ulcerative
agents cause a depletion of the catecholamine
store, an increase in the acetylcholine level in
the gastric mucosa, a release of serotonin and
histamine, an increase in the plasma corticoste-
roid and vasopressin levels, disturbances in the
ion-exchanging biopolymer function and electro-
lyte equilibrium. Such a wide range of effects
evidences the extent of difculties encountered
during pharmacological evaluation of the anti-
ulcerative activity of new substances in various
models of experimental gastric and duodenal ul-
cers (Ok a b e et al., 1978; Ok a b e et al., 1992; Ok a b e
et al., 1997).
Both the experiments conducted in the course
of the present study and the literature data on
propolis composition suggest that it has many
targets, each of them playing a specic role, in-
dependently of the mechanism of experimental
peptic ulcer development. It is also probable that
many known and unknown propolis properties
are complementary to each other and in this way
they prevent the ulcerative process development
and induce regenerative processes. The obtained
results indicate that the observed antiulcerative
effects of propolis may consist in the multifunc-
tional biological role of this natural product col-
lected by bees and used in Polish folk medicine
for ages.
High pharmacological activity of propolis
which allows it to exert a signicant protective
effect on experimentally induced gastric and
duodenal ulcers justies both further detailed
pharmacological studies and the need for closer
attention given by clinical centres to this bee
product.
ACKNOWLEDGMENTS
The authors would like to thank Mrs. Teresa Do-
brut for her technical assistance.

42 Czarnecki and Librowski
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