in vitro antibacterial activity of propolis extracts on 12 different bacteria in conditions of 3 various ph

20 Pages·2010·0.19 MB·English

Arch. Biol. Sci., Belgrade, 62 (4), 915-934, 2010 DOI:10.2298/ABS1004915I
IN VITRO ANTIBACTERIAL ACTIVITY OF PROPOLIS EXTRACTS ON 12 DIFFERENT BACTERIA
IN CONDITIONS OF 3 VARIOUS PH VALUES
S. IVANČAJIĆ , I. MILEUSNIĆ and DESANKA CENIĆ-MILOŠEVIĆ
Faculty of Stomatology, 26000 Pančevo, Serbia
Abstract - This research investigated the effects of propolis extracted by 5 different solvents and aged for 7 days on
twelve species of bacteria classified into four groups according to their pathogenicity in slightly acidic (pH=6), neutral
(pH=7) and slightly alkaline (pH=8) environments. Propolis extracted by the examined solvents had antibacterial
effects. The strongest effects on the growth of all tested microorganisms, except on the bacteria of the Salmonella genus,
regardless of the pH value of the environment, were exerted by propolis extracted by ether, acetone, toluol and
chloroform. In some cases the antibacterial action of propolis was best in a slightly acidic environment (pH=6).
Key words: Bacterial cultures, pH of the environment, propolis, solvents.
UDC 638.135:615.4.51:615.281
roethane, chloroform, carbon tetrachloride, diethyl
INTRODUCTION
ether, benzol) solvents are used. Today, ethanol is
generally the solvent used in the process of extrac-
Propolis is a rubbery, sticky, brown, thermoplastic
tion of propolis and all published data show the
resin collected by honey-bees from tree buds. Ho-
effects of propolis extracts dissolved in ethanol. Sin-
ney bees use propolis in their hives as a universal
ce ethanol belongs to the group of less polar sol-
means of repairing crevices, as a surface cover, har-
vents, when used as an extractant only less polar ac-
dener and preservative. It is probably also used as a
tive substances are extracted, while flavonoids are
repellent since it is applied inside the beehive and
extracted in minimal quantities. Using nonpolar
around its entrance. The term propolis originates
solvents, nonpolar flavonoids can better be ex-
from the Greek words pro – meaning before/in
tracted from the basic substrate, leading to an in-
front and polis – meaning town (Jankovic, 1968)
crease of their concentration in the final prepara-
and denotes the fact that bees use propolis to con-
tion. Pharmacologically active constituents of pro-
struct the entrance to the beehive – “town”. It is
polis have been discovered in fractions that are so-
also known as “bee glue”.
luble in solvents such as ethanol. Several large
Propolis has bactericidal and fungicidal properties classes of active substances have been identified in a
and it is used as an alternative treatment for infections. variety of constituents of propolis. Among these
The wide range of action of propolis on various are: flavones, flavonoles and flavanones (known
microorganisms is the result of the combined activities under the collective name of flavonoids), various
of flavonoids and aromatic compounds. phenols and aromatics. Flavonoids represent a large
group of natural pigments and active compounds of
In principle, active components of a natural herbal origin. All flavonoids have two benzene rings
preparation can be separated by solvents in the pro- linked by a triple bond. The most significant phe-
cess of extraction. Solvents of different polarity may nols are: cinnamyl alcohol, cinnamic acid, vanillin,
be used as extractants. For this purpose polar (wa- benzyl alcohol, benzoic acid and caffeic and ferulic
ter, glycerol, methanol), less polar (ethanol, propyl acids (which are derivatives of cinnamic acid with
alcohol, acetone, and others) and nonpolar (dichlo- an extra hydroxy and/or methyloxy group). Propo-
915
916 S. IVANČAJIĆ ET AL.
lis also contains rare compounds such as phenol University of Belgrade, were used in this research.
triglycerides (Popravko et al., 1983), pterostilbene The bacterial species were chosen according to the
and eugenol and phenethyl ester of caffeic acid. The frequency that they were used in various researches
principal pharmacological constituents of propolis and also according to the frequency of infections in
are natural antibiotics – flavonoids which exert humans. Pure cultures of bacterial species be-
most of the healing properties of propolis. The anti- longing to four different groups, according to the
microbial activity of propolis has been well inves- classification published in Bergey’s Manual of
tigated (Ghisalberti, 1979; Grange and Davey, Determinative Bacteriology (Tortora et al., 2004)
1990). Flavon pinocembrin affects numerous bacte- were used in this research. This classification is one
ria, fungi and molds and it is, together with galan- of the best known and most accepted among
gin, 3-acetyl pinobanksin and caffeic and ferulic microbiologists.
acid, probably responsible for many of the biolo-
gical activities of propolis (Hegazi and Abd El Ha- I group - bacteria banal: Proteus morgani (mor-
dy, 2001; Hegazi and Abd El Hady, 2002a; Hegazi ganella), Streptococcus faecalis, Achromobacter –
and Abd El Hady, 2002b; Popova et al., 2004). Acinetobacter, Sarcina lutea and Escherichia coli.
During the physiological processes in an organism II group - opportunistic pathogenic bacteria:
the mechanism of homeostasis maintains the pH value Aeromonas hydrophila, Salmonella typhimurium
of the internal environment around the neutral level. and Bacillus subtilis.
However, during pathophysiological processes, the che-
III group – infectious pathogenic bacteria:
mical reactions that take place decrease the level of pH,
Salmonella gallinarum and Salmonella cholerae.
making the affected environment slightly acidic.
IV group - exotoxic pathogenic bacteria:
The principal hypothesis of this study was that
Staphylococcus aureus and Bacillus cereus.
the bactericidal and antimicrobial effects of propo-
lis vary depending on: the type of solvent used du-
To ensure the homogeneity of the solution, only
ring the extraction, bacterial species and acidity of
propolis from one colony and a fixed time period
the environment.
was used in this study.
The aim of this study was to define which pre-
Five solvents were used to extract the active
paration of propolis had most antimicrobial activity
ingredients in propolis: ethanol (C2H5OH), ether
so that the way propolis is used could be improved.
(C2H5-O-C2H5), acetone ((CH3)2CO), chloro-
The bactericidal effect of propolis extracted by dif-
form (CHCl3) and toluol (C6H5CH3). The extrac-
ferent solvents (ethanol, ether, acetone, toluol and
ted propolis was aged for 7 days and used for expe-
chloroform) aged for 7 days on cultures of 12 diffe-
riments that were performed with all test microor-
rent species of bacteria (Morganella morgani, Strep-
ganisms. Extraction was performed in the following
tococcus faecalis, Achromobacter, Sarcina lutea,
manner, regardless of the type of solvent. The mix
Escherichia coli, Aeromonas hydrophila, Salmonella
of solvent and water (volume ratio from 60:40 up to
typhimurium, Bacillus subtilis, Salmonella galli-
96:4) was poured into a mixer with a double contai-
narum, Salmonella cholerae, Staphylococcus aureus,
ner (used for cooling purposes). Then, when etha-
Bacillus cereus) in varying (pH=6, pH=7 and pH=8)
nol was used, 500 ml of ethanol was added to 150 g
environment conditions, was investigated.
of propolis. The extraction process lasted for 48 h,
after which 465 ml of filtrate were obtained with a
MATERIAL AND METHODS specific weight of 0.845. The weight of the precipi-
tate was 130 g, the temperature of the water bath
Standardized pure cultures of bacterial strains was 65°C and the steaming time was 20 min. The
procured from the Faculty of Veterinary Medicine, weight of the propolis extract obtained was 60 g.
IN VITRO ANTIBACTERIAL ACTIVITY OF PROPOLIS EXTRACTS 917
When ether was used for the extraction, 500 ml of this experiment agar (obtained from Institute of
ether was added to 150 g of propolis. The extraction Virology, Vaccines and Sera “Torlak”, Belgrade,
process lasted for 48 h, after which 360 ml of filtrate Serbia) was used as the growth medium. The agar
were obtained with a specific weight of 0.782. The consisted of peptone, beef extract, potassium phos-
weight of the precipitate was 160 g, the temperature phate and agar in standardized proportions. This
of the water bath was 40°C, and the steaming time medium altogether consisted of peptone, beef ex-
was 10 min. The weight of the propolis extract was tract, potassium phosphate and agar in a standar-
80 g. When acetone was used, 500 ml of acetone dized proportion. 41.3 g of agar powder was sus-
was added to 150 g of propolis. The extraction las- pended in 1000 ml of cold distilled water and left
ted 48 h yielding 450 ml of filtrate with a specific for 15 min. The medium was then carefully heated
weight of 0.842. The weight of the precipitate was to boiling point until completely dissolved and pou-
130 g, the temperature of the water bath was 55°C, red into bottles and sterilized in an autoclave for 15
and the steaming time was 30 min. The weight of min at 120°C. After sterilization, the pH value of
the propolis extract was 80 g. When extraction was the medium was adjusted to 6, or 7, or 8, by the
done by chloroform, 500 ml of chloroform was addition of either 0.1 mol of hydrochloric acid
added to 150 g of propolis. The extraction took solution or 0.1 mol of sodium hydroxide solution.
place for 48 h, yielding 500 ml of filtrate with a The melted nutritious base was divided into several
specific weight of 1.350. The weight of the precipi- parts. After cooling down to 45-50°C each part was
tate was 120 g, the temperature of the water bath cultivated by the culture of one test microorganism.
was 55°C and the steaming time was 45 min. The
weight of the propolis extract was 150 g. When ex- The cultures of all 12 test microorganisms were
traction was done by toluol, 500 ml of toluol was diluted ten-fold (down to 0.1 mol) by the addition
added to 150 g of propolis. The extraction process of sterilized physiological solution and cultivated
lasted for 48 h, yielding 486 ml of the filtrate with a on 100 ml of nutritious agar medium that had been
specific weight of 0.865. The weight of the precipi- melted and cooled to 45-50°C. In such a manner
tate was 160 g, the temperature of the water bath nutritious agar having three different pH values
was 65°C, and the steaming time was 15 min. The could be cultivated with each of the 12 species of
weight of the propolis extract was 40 g. test microorganisms. The cultivated agar was pou-
red into plastic, sterile Petri dishes, volume 100 ml,
Cooling was done at 278 – 288K for 30 min and so that the thickness of the base was 2 mm. Three
mixing at the speed of 20 m/sec. The resulting suspen- holes, 10 mm in diameter, were drilled in the set
sion was centrifuged three times in succession. The ob- agar in each Petri dish.
tained extract was a clear liquid, dark brown in color.
The antimicrobial activity of the propolis
Test microorganisms were cultivated in a nutri- samples was investigated by the method of growth
tious broth (obtained from Institute of Virology, inhibition of the chosen test microorganism in the
Vaccines and Sera “Torlak”, Belgrade, Serbia).The culture medium. Various propolis extracts were
broth contained peptones, beef extract, sodium placed in the holes made in the cultivated growth
chloride and potassium phosphate in a standardi- medium from which it could freely diffuse into the
zed proportion. The dissolved base broth was hea- environment. The growth of the investigated bacte-
ted to boiling point until completely dissolved. The rium was inhibited in the diffusion zone according
dissolved base, having a pH value of 7.3 was poured to its sensitivity to propolis. The width of this inhi-
into test tubes and sterilized in an autoclave for 15 bition zone showed the degree of sensitivity of the
min at 120°C. Broths cultivated by test microor- investigated bacterium to propolis, ranging from a
ganisms were incubated for 24 h at 30°C. Each test narrow or nonexistent inhibition zone, in the case
microorganism was re-cultivated in this manner of resistance, to a wider zone, corresponding to cer-
three days in succession before the experiment. In tain degrees of sensitivity.
918 S. IVANČAJIĆ ET AL.
Table 1. Activity of the propolis solution aged for 7 days on Morganella morgani in slightly acidic (pH=6), neutral (pH=7) and
slightly alkaline (pH=8) cultures.
Morganella morgani
ethanol ether acetone toluol chloroform
pH=6 7.3 8 7.6 9* 6.3
pH=7 6.6 6.6 7 6.3 5.3
pH=8 6.3 6.3 4.3 5 6.3
SE pH=6 ±0.14 ±0.42 ±0.32 ±0.24 ±0.15
SE pH=7 ±0.35 ±0.27 ±0.5 ±0.25 ±0.27
SE pH=8 ±0.22 ±0.51 ±0.27 ±0.43 ±0.17

10
9
8
7
6
pH=6
5 pH=7
pH=8
4
3
2
1
0
ethanol ether acetone toluol chloroform
Morganella morgani

Fig. 1. Activity of the propolis solution aged for 7 days on Morganella morganiin slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
Measuring procedure for antimicrobial activity was measured from the margin of the hole to its ou-
of propolis ter border.
Propolis extracts were heated in a water bath at The value, expressed in millimeters, was the
50°C until a semi-liquid consistency was reached. mean value of measurements around all three holes
Each sample was subsequently poured into holes in one Petri dish.
made in the nutritious agar base. The prepared ho-
les were completely filled by the propolis extracts. Since there more than one statistical sample
The cultures were then incubated for 2 h at 4°C, (propolis extracted in 5 types of solvents at 3 pH
followed by 18 h at 30°C. The width of the inhibi- values), Student T-test according to Bonferroni
tion zone of growth of the tested microorganism (Steel RGD et al., 1960) was used to determine the
IN VITRO ANTIBACTERIAL ACTIVITY OF PROPOLIS EXTRACTS 919
level of differences between the arithmetical means statistically significant (p<0.05) stronger effect of
of the samples’ modification. propolis extracted by chloroform in cultures with
pH=6 and pH=8 on bases with pH=6 and 8 than in
cultures with a pH value of 7.
RESULTS
2. Streptococcus faecalis
All results have been statistically processed and
shown in tables and figures. Each investigated para-
Table 2 and Fig. 2 show mean values of the inhibi-
meter is represented by the mean value and statis-
tion zones of propolis aged for 7 days on bacterial
tical significance, separately marked.
cultures of Streptococcus faecalis in slightly acidic
(pH=6), neutral (pH=7) and slightly alkaline
Results of the inhibitory effect
(pH=8) cultures.
of 5 preparations of propolis in cultures with a pH
value of 6, 7 and 8 on bacteria from group I
The results showed that the effect of propolis
extracted by acetone and applied in the growth me-
1. Morganella morgani
dium with a pH value of 6 was most distinct and
statistically significantly (p<0.01) stronger than the
Table 1 and Fig. 1 show mean values of the inhi-
inhibitory activity of other types of propolis on the
bition zones of propolis aged for 7 days on bacterial
growth of Streptococcus faecalis.
cultures of Morganella morgani in slightly acidic
(pH=6), neutral (pH=7) and slightly alkaline
The results of the inhibitory effect of propolis
(pH=8) cultures.
extracted by various types of solvents on Strepto-
It was clear that propolis extracted in any sol- coccus faecalis depending on the pH value of cul-
vent had a significant inhibitory effect on the tures showed that there was a statistically significant
growth of Morganella morgani. The propolis extrac- (p<0.001) stronger effect of propolis extracted by
ted in toluol at pH=6 exhibited the most distinct, ethanol in cultures with pH=6 than in cultures with
statistical significant (p<0.001) effect, and the pro- a pH value of 8 and a statistically significantly
polis extracted by acetone, at pH=8, had the lowest greater effect (p<0.01) than in cultures with a pH
effect on the growth of Morganella morgani value of 7. There was a statistically significant
(p<0.001), (p<0.001) stronger effect of propolis extracted by
ether in cultures with pH=6 than in cultures with a
The results of the inhibitory effect of propolis pH value of 7 and 8. There was a statistically
extracted by various types of solvents on Morga- significant (p<0.001) stronger effect of propolis
nella morgani depending on the pH value of cultu- extracted by acetone in cultures with pH=6 than in
res showed that there was a statistically significant cultures with a pH value of 7 and 8. There was a
(p<0.05) stronger effect of propolis extracted by statistically significant (p<0.05) stronger effect of
ethanol in cultures with pH=6 than in cultures with propolis extracted by toluol in cultures with pH=8
a pH value of 8. There was a statistically significant than in cultures with a pH value of 6. There was a
(p<0.05) stronger effect of propolis extracted by statistically significant (p<0.001) stronger effect of
ether in cultures with pH=6 than in cultures with a propolis extracted by chloroform in cultures with
pH value of 7 and 8. There was a statistically pH=6 than in cultures with a pH value of 7 and 8.
significant (p<0.001) stronger effect of propolis ex-
tracted by acetone in cultures with pH=6 than in 3. Achromobacter
cultures with a pH value of 8. There was a statisti-
cally significant (p<0.001) stronger effect of pro- Table 3 and Fig. 3 show mean values of the
polis extracted by toluol in cultures with pH=6 than inhibition zones of propolis aged for 7 days on
in cultures with a pH value of 7 and 8. There was a bacterial cultures of Achromobacter in slightly aci-
920 S. IVANČAJIĆ ET AL.

Table 2. Activity of the propolis solution aged for 7 days on Streptococcus faecalis in slightly acidic (pH=6), neutral (pH=7) and
slightly alkaline (pH=8) cultures.
Streptococcus faecalis
pH=6 pH=6 pH=6 pH=6 pH=6 pH=6
pH=7 pH=7 pH=7 pH=7 pH=7 pH=7
pH=8 pH=8 pH=8 pH=8 pH=8 pH=8
SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6
SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7
SE pH=8 SE pH=8 SE pH=8 SE pH=8 SE pH=8 SE pH=8

9
8
7
6
pH=6
5
pH=7
4
pH=8
3
2
1
0
ethanol ether acetone toluol chloroform
Streptococcus faecalis

Fig. 2. Activity of the propolis solution aged for 7 days on Streptococcus faecalisin slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
dic (pH=6), neutral (pH=7) and slightly alkaline 4. Sarcina lutea
(pH=8) cultures.
Table 4 and Fig. 4 show mean values of the inhi-
All types of propolis had a distinct inhibitory bition zones of propolis aged for 7 days on bacterial
activity on the growth of Achromobacter. However cultures of Sarcina lutea in slightly acidic (pH=6),
propolis extracted by toluol had a statistically neutral (pH=7) and slightly alkaline (pH=8)
significantly (p<0.001) greater inhibitory activity cultures.
than any other type of propolis, but without
statistical significance as regards to the pH of the The results showed that all preparations of
medium. propolis had a moderate inhibitory activity on the
IN VITRO ANTIBACTERIAL ACTIVITY OF PROPOLIS EXTRACTS 921

Table 3. Activity of the propolis solution aged for 7 days on Achromobacterin slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
Achromobacter

pH=6 pH=6 pH=6 pH=6 pH=6 pH=6
pH=7 pH=7 pH=7 pH=7 pH=7 pH=7
pH=8 pH=8 pH=8 pH=8 pH=8 pH=8
SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6
SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7

12
10
8
pH=6
6 pH=7
pH=8
4
2
0
ethanol ether acetone toluol chloroform
Achromobacter

Fig. 3. Activity of the propolis solution aged for 7 days on Achromobacter in slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
growth of Sarcina lutea, while there was a sta- with a pH value of 8. There was a statistically
tistically significantly (p<0.01) stronger inhibitory significant (p<0.05) stronger effect of propolis
effect of propolis extracted by acetone and toluol in extracted by ether in cultures with pH=6 than in
cultures with a pH value of 6 and 7. cultures with a pH value of 7. There was a
statistically significant (p<0.001) stronger effect
Results of the inhibitory effect of propolis of propolis extracted by toluol in cultures with
extracted by various types of solvents on Sarcina pH=6 and 7 than in cultures with a pH value of
lutea depending on the pH value of cultures 8. Propolis extracted by acetone or chloroform
showed that there was a statistically significant did not show any significant inhibitory activity
(p<0.01) stronger effect of propolis extracted by on Sarcina lutea regardless of the pH value of
ethanol in cultures with pH=6 than in cultures the medium.
922 S. IVANČAJIĆ ET AL.
Table 4. Activity of the propolis solution aged for 7 days on Sarcina lutea in slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
Sarcina lutea

pH=6 pH=6 pH=6 pH=6 pH=6 pH=6
pH=7 pH=7 pH=7 pH=7 pH=7 pH=7
pH=8 pH=8 pH=8 pH=8 pH=8 pH=8
SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6
SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7
* statistically significant (p<0.01)

12
10
8
pH=6
6 pH=7
pH=8
4
2
0
ethanol ether acetone toluol chloroform
Sarcina lutea

Fig. 4. Activity of the propolis solution aged for 7 days on Sarcina luteain slightly acidic (pH=6), neutral (pH=7) and slightly alkaline
(pH=8) cultures.
5. Escherichia coli effect than other extracts of propolis, in a slightly
acidic environment (pH=6).
Table 5 and Fig. 5 show mean values of the
inhibition zones of propolis aged for 7 days on Results of the inhibitory effect of propolis
bacterial cultures of Escherichia coli in slightly extracted by various types of solvents on Escherichia
acidic (pH=6), neutral (pH=7) and slightly alkaline coli depending on the pH value of cultures showed
(pH=8) cultures. that there was a statistically significant (p<0.01)
stronger effect of propolis extracted by ethanol in
The results showed that inhibitory activity of all cultures with pH=6 than in cultures with a pH value
extracts of propolis on the growth of Escherichia of 7 and a statistically significantly (p<0.001)
coli was distinct, and that propolis aged 7 days greater inhibitory effect than in cultures with a pH
extracted by toluol and chloroform had a sta- value of 8. There was a statistically significant
tistically significantly (p<0.001) stronger inhibitory (p<0.05) stronger effect of propolis extracted by
IN VITRO ANTIBACTERIAL ACTIVITY OF PROPOLIS EXTRACTS 923
Table 5. Activity of the propolis solution aged for 7 days on Escherichia coliin slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.
Escherichia coli

pH=6 pH=6 pH=6 pH=6 pH=6 pH=6
pH=7 pH=7 pH=7 pH=7 pH=7 pH=7
pH=8 pH=8 pH=8 pH=8 pH=8 pH=8
SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6
SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7
* statistically significant (p<0.01)

14
12
10
8 pH=6
pH=7
6 pH=8
4
2
0
ethanol ether acetone toluol chloroform
Escherichia coli
Fig. 5. Activity of the propolis solution aged for 7 days on Escherichia coli in slightly acidic (pH=6), neutral (pH=7) and slightly
alkaline (pH=8) cultures.

ether in cultures with pH=6 than in cultures with Results of the inhibitory effect of 5 preparations of
pH values of 7 and 8. Propolis extracted by acetone propolis in cultures with pH values of 6, 7 and 8 on
did not show any significant inhibitory activity on bacteria from group II
Escherichia coli regardless of the pH value of the
medium. There was a statistically significant 1. Aeromonas hydrophila
(p<0.001) stronger effect of propolis extracted by
toluol in cultures with pH=6 than in cultures with Table 6 and Fig. 6 show mean values of the
pH values of 7 and 8. There was a statistically inhibition zones of propolis aged for 7 days on
significant (p<0.001) stronger effect of propolis bacterial cultures of Aeromonas hydrophila in
extracted by chloroform in cultures with pH=6 than slightly acidic (pH=6), neutral (pH=7) and slightly
in cultures with pH values of 7 and 8. alkaline (pH=8) cultures.
924 S. IVANČAJIĆ ET AL.
Table 6. Activity of the propolis solution aged for 7 days on Aeromonas hydrophila in slightly acidic (pH=6), neutral (pH=7) and
slightly alkaline (pH=8) cultures.
Aeromonas hydrophila

pH=6 pH=6 pH=6 pH=6 pH=6 pH=6
pH=7 pH=7 pH=7 pH=7 pH=7 pH=7
pH=8 pH=8 pH=8 pH=8 pH=8 pH=8
SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6 SE pH=6
SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7 SE pH=7
* statistically significant (p<0.01)

9
8
7
6
pH=6
5
pH=7
4
pH=8
3
2
1
0
ethanol ether acetone toluol chloroform
Aeromonas hydrofila
Fig. 6. Activity of the propolis solution aged for 7 days on Aeromonas hydrophila in slightly acidic (pH=6), neutral (pH=7) and
slightly alkaline (pH=8) cultures.

All propolis preparations had a moderate ted by ethanol in cultures with pH=6 than in
inhibitory activity on the growth of Aeromonas cultures with a pH value of 7. There was a sta-
hydrophila. Statistically significantly greater tistically significant (p<0.001) stronger effect of
(p<0.01) inhibitory activity was exhibited by pro- propolis extracted by ether in cultures with pH=6
polis extracted by ether and by acetone in cultures than in cultures with pH values of 7 and 8. There
with a pH value of 6 compared to the other extracts was a statistically significant (p<0.001) stronger
used. effect of propolis extracted by acetone in cultures
with pH=6 than in cultures with pH values of 7 and
Results of the inhibitory effect of propolis 8. There was a statistically significant (p<0.001)
extracted by various types of solvents on Aero- stronger effect of propolis extracted by toluol in
monas hydrophila depending on the pH value of cultures with pH=6 than in cultures with pH values
cultures showed that there was a statistically signi- of 7 and 8. Propolis extracted by chloroform did not
ficant (p<0.001) stronger effect of propolis extrac- show any significant inhibitory activity on Aeromo-